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1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) in Efficient Amide Bond Formation for Peptide Synthesis: A Comprehensive Review

Abstract: 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) is a strong, non-nucleophilic organic base widely employed in organic synthesis. This article provides a comprehensive overview of its application in efficient amide bond formation, particularly in the context of peptide synthesis. We delve into the reaction mechanisms, advantages, and limitations of DBU-mediated amide bond formation, compare it with other commonly used bases, and highlight its specific roles in various peptide synthesis strategies. The discussion encompasses the influence of reaction conditions, protecting group selection, and substrate structure on reaction efficiency. Furthermore, the article outlines the product parameters of DBU and provides examples from the literature showcasing its versatility in both solution-phase and solid-phase peptide synthesis.

1. Introduction

Amide bond formation is a fundamental reaction in organic chemistry, crucial for the synthesis of peptides, proteins, pharmaceuticals, and various other biologically active compounds. Peptide synthesis, in particular, relies heavily on efficient and selective amide bond formation to link amino acid building blocks. Several coupling reagents and reaction conditions have been developed to facilitate this process. Among these, the use of bases plays a critical role in activating the carboxyl component and neutralizing the acidic byproducts generated during the coupling reaction. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) has emerged as a versatile and widely used base in peptide synthesis due to its strong basicity, non-nucleophilic character, and relatively low cost.

2. Properties of DBU

DBU is a bicyclic guanidine derivative with the chemical formula C9H16N2 and a molecular weight of 152.23 g/mol. Its structure features a highly delocalized positive charge upon protonation, contributing to its strong basicity and reduced nucleophilicity.

DBU is commercially available in various grades, including anhydrous forms, ensuring minimal water interference in sensitive reactions. It is typically stored under inert atmosphere to prevent degradation by atmospheric carbon dioxide or moisture.

3. Mechanism of Amide Bond Formation with DBU

DBU facilitates amide bond formation through several mechanisms, depending on the specific coupling reagent and reaction conditions employed. Generally, DBU acts as a base to:

• Deprotonate the carboxyl group: DBU abstracts a proton from the carboxylic acid of the activated amino acid derivative, forming a carboxylate anion. This anion is a better nucleophile and more readily attacks the electrophilic amine component.
• Neutralize acidic byproducts: Many coupling reactions generate acidic byproducts (e.g., HOAt, HOBt from HATU or HOBt activation strategies). DBU neutralizes these acids, preventing them from protonating the amine component and hindering the coupling reaction.
• Promote specific coupling reagent activation: In some cases, DBU is involved in the activation of the coupling reagent itself, facilitating the formation of the active ester or other reactive intermediate.

Example Mechanism (HOBt/HBTU Activation):

1. The carboxylic acid reacts with HOBt or HBTU to form an active ester (e.g., HOBt ester).
2. DBU deprotonates the carboxylic acid and/or HOBt/HBTU reagent, promoting the formation of the active ester.
3. DBU neutralizes the released acid (HOBt or HBTU).
4. The amine component attacks the active ester, forming the amide bond and releasing HOBt.

4. Advantages of DBU in Peptide Synthesis

DBU offers several advantages as a base in peptide synthesis:

• Strong Basicity: Its high pKa value ensures efficient deprotonation of the carboxylic acid, promoting rapid and complete coupling reactions.
• Non-Nucleophilicity: DBU is a sterically hindered base, minimizing its participation in unwanted side reactions, such as epimerization or racemization. This is crucial for maintaining the stereochemical integrity of the chiral amino acid building blocks.
• Solubility: DBU is soluble in a wide range of organic solvents, including DMF, DCM, and acetonitrile, which are commonly used in peptide synthesis.
• Commercial Availability and Cost-Effectiveness: DBU is readily available from numerous chemical suppliers at a reasonable cost, making it an attractive choice for both research and industrial applications.
• Compatibility with Various Protecting Groups: DBU is generally compatible with common protecting groups used in peptide synthesis, such as Boc, Fmoc, and Cbz. However, careful consideration is required depending on the specific protecting group strategy employed.
• Facilitates Racemization-Free Coupling: Compared to more nucleophilic bases, DBU is less likely to induce racemization at the α-carbon of the amino acids, preserving the desired stereochemistry of the peptide product.

5. Limitations and Considerations

Despite its advantages, DBU also has some limitations that need to be considered:

• Potential for β-Elimination: Under strongly basic conditions, DBU can promote β-elimination reactions, particularly in amino acids containing β-substituents (e.g., serine, threonine). Careful optimization of reaction conditions is required to minimize this side reaction.
• Sensitivity to Moisture and Carbon Dioxide: DBU is hygroscopic and can react with atmospheric carbon dioxide, leading to the formation of carbonates. Anhydrous conditions and inert atmosphere are recommended for optimal results.
• Base-Catalyzed Deprotection: In some cases, DBU can catalyze the removal of certain protecting groups, leading to undesired side reactions. This is particularly relevant when using base-labile protecting groups.
• Influence of Solvent: The solvent used in the reaction can significantly influence the basicity and reactivity of DBU. Protic solvents can reduce its basicity through hydrogen bonding.
• Optimization Required: The optimal concentration of DBU, reaction temperature, and reaction time need to be optimized for each specific coupling reaction.

6. Comparison with Other Commonly Used Bases in Peptide Synthesis

Several other bases are commonly used in peptide synthesis, each with its own advantages and disadvantages. A comparison with some of the most prevalent bases is presented below:

7. Applications of DBU in Peptide Synthesis

DBU finds widespread application in both solution-phase and solid-phase peptide synthesis (SPPS).

7.1. Solution-Phase Peptide Synthesis

In solution-phase synthesis, DBU is commonly used as a base to neutralize acidic byproducts generated during the coupling reaction and to facilitate the activation of the carboxyl component. It is particularly useful in coupling reactions involving sterically hindered amino acids or when using coupling reagents prone to racemization.

• Example 1: Synthesis of a dipeptide using HBTU/HOBt coupling: A protected amino acid (e.g., Fmoc-Ala-OH) is activated with HBTU and HOBt in the presence of DBU in DMF. The activated amino acid is then coupled with a protected amino acid ester (e.g., H-Val-OMe) to form the dipeptide.

  Fmoc-Ala-OH + HBTU + HOBt + DBU  -->  Fmoc-Ala-O(HOBt)
  Fmoc-Ala-O(HOBt) + H-Val-OMe  -->  Fmoc-Ala-Val-OMe

• Example 2: Macrolactamization: DBU can be used to promote the intramolecular cyclization of linear peptides to form cyclic peptides (macrolactams). The carboxyl group is activated in situ, and DBU facilitates the cyclization by deprotonating the amine component. [Reference 1]

7.2. Solid-Phase Peptide Synthesis (SPPS)

DBU is frequently employed in Fmoc-based SPPS, particularly in the following applications:

• Neutralization of Acidic Salts: The N-terminal amine of the resin-bound amino acid is often protected as a hydrochloride or trifluoroacetate salt. DBU is used to neutralize these salts prior to coupling with the next amino acid.
• Activation of Coupling Reagents: DBU can be used in conjunction with various coupling reagents, such as HATU, HCTU, and DIC/Oxyma, to promote efficient amide bond formation on the solid support. [Reference 2]
• Removal of Fmoc Protecting Group: DBU is a key component in the standard Fmoc deprotection protocols. A solution of DBU in DMF is used to remove the Fmoc protecting group from the N-terminal amine of the resin-bound peptide. This is a crucial step in each cycle of Fmoc-based SPPS. Typically, a mixture of DBU and piperidine is used. Piperidine acts as a scavenger to trap dibenzofulvene, the byproduct of Fmoc deprotection.
• Cyclization on Resin: DBU can be used to promote on-resin cyclization of peptides. [Reference 3]

7.3. Specific Examples from Literature

• Example 1: DBU-catalyzed Peptide Coupling with Vinyl Azides: A novel method for peptide coupling using vinyl azides as carboxyl-activating agents, catalyzed by DBU, has been reported. This method allows for efficient peptide bond formation under mild conditions. [Reference 4]

• Example 2: DBU in the Synthesis of β-Peptides: DBU has been used in the synthesis of β-peptides, which are oligomers of β-amino acids. Its non-nucleophilic character is advantageous in preventing side reactions during the coupling of these modified amino acids. [Reference 5]

• Example 3: DBU in the Synthesis of Depsipeptides: DBU is employed in the synthesis of depsipeptides, which contain both amide and ester bonds. The presence of the ester bond requires careful selection of reaction conditions to avoid ester hydrolysis. DBU, with its controlled basicity, allows for selective amide bond formation without compromising the ester functionality.

8. Factors Influencing Amide Bond Formation with DBU

The efficiency of amide bond formation using DBU is influenced by several factors:

• Solvent: The choice of solvent can significantly impact the reaction rate and yield. Polar aprotic solvents, such as DMF and NMP, are generally preferred as they enhance the solubility of the reactants and facilitate the deprotonation of the carboxylic acid.
• Temperature: The reaction temperature can affect both the rate of amide bond formation and the extent of side reactions. Lower temperatures are often preferred to minimize racemization, while higher temperatures may be necessary to overcome steric hindrance.
• Concentration of DBU: The optimal concentration of DBU needs to be carefully optimized. An insufficient amount of DBU may result in incomplete deprotonation, while an excessive amount may promote side reactions.
• Coupling Reagent: The choice of coupling reagent plays a crucial role in the success of the reaction. DBU is compatible with a wide range of coupling reagents, including carbodiimides (DIC, DCC), uronium salts (HBTU, HATU), and phosphonium salts (PyBOP).
• Protecting Groups: The protecting groups used to protect the amino and carboxyl functionalities can influence the reaction rate and selectivity. The protecting groups should be stable under the reaction conditions and readily removable after the coupling reaction.
• Steric Hindrance: Sterically hindered amino acids may require longer reaction times and higher concentrations of DBU to achieve complete coupling.
• Additives: Additives such as HOBt and HOAt can enhance the efficiency of the coupling reaction by suppressing racemization and improving the solubility of the reactants.

9. Conclusion

DBU is a valuable and versatile base for efficient amide bond formation in peptide synthesis. Its strong basicity, non-nucleophilic character, and compatibility with various coupling reagents and protecting groups make it a widely used reagent in both solution-phase and solid-phase peptide synthesis. While DBU offers several advantages, careful consideration of its limitations and optimization of reaction conditions are essential for achieving high yields and minimizing side reactions. Understanding the factors that influence amide bond formation with DBU allows for the rational design of peptide synthesis strategies and the efficient production of complex peptide molecules. Future research efforts may focus on developing modified DBU derivatives with enhanced properties, such as improved solubility or reduced propensity for β-elimination, further expanding its utility in peptide and organic synthesis.

10. References

1. Schmidt, U.; Langner, J. J. Org. Chem. 1995, 60, 7054-7057.
2. Carpino, L. A. J. Am. Chem. Soc. 1993, 115, 4397-4398.
3. Bogdanowicz, M. J.; Sabat, M.; Rich, D. H. J. Org. Chem. 2003, 68, 5626-5636.
4. Zhang, L.; et al. Org. Lett. 2018, 20, 7896-7900.
5. Seebach, D.; et al. Helv. Chim. Acta 1996, 79, 913-941.

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