The Use of 9-fluorenylmethoxycarbonyl in peptide synthesis

9-Fluorenylmethoxycarbonyl (Fmoc) is a widely used protective group in peptide synthesis, particularly in solid-phase peptide synthesis (SPPS). Fmoc protects the amino group of amino acids during peptide chain elongation, preventing unwanted side reactions. 

Key Features and Advantages of Fmoc

1. Base-Labile Protection:
   • Fmoc is removed using mild basic conditions, typically 20% piperidine in dimethylformamide (DMF). This mild deprotection prevents side reactions that might occur under harsher conditions.
   • This base-labile nature contrasts with t-butyloxycarbonyl (Boc), another common protecting group, which is acid-labile.
2. Compatibility with Acid-Labile Resins:
   • Fmoc strategy is often used with acid-labile resins, such as Wang or Rink amide resins. After completing the peptide synthesis, the peptide-resin bond and side-chain protecting groups are cleaved using a strong acid such as trifluoroacetic acid (TFA).
3. Orthogonal Protection Strategy:
   • The Fmoc/t-Boc orthogonal protection strategy allows for selective removal of protecting groups without affecting the peptide or resin linkage. Fmoc is removed under basic conditions, while Boc is removed under acidic conditions, providing flexibility in protecting different functional groups on amino acids.
4. Efficient Deprotection Monitoring:
   • The deprotection of Fmoc can be monitored visually due to the release of the dibenzofulvene chromophore, which absorbs strongly at 290-300 nm. This allows real-time monitoring of the deprotection step and helps ensure complete removal.

Steps Involving Fmoc in SPPS

1. Amino Acid Attachment:
   • The first amino acid (with Fmoc-protected amino group) is attached to the resin via its carboxyl group. The attachment is facilitated by activating the carboxyl group using reagents like HBTU, HATU, or DIC.
2. Deprotection Cycle:
   • The Fmoc group is removed using 20% piperidine in DMF, exposing the free amino group for subsequent coupling.
   • Deprotection is typically performed twice to ensure complete removal of the Fmoc group.
3. Coupling Reaction:
   • The next Fmoc-protected amino acid is activated and coupled to the free amino group of the growing peptide chain on the resin.
   • This cycle of deprotection and coupling is repeated until the desired peptide sequence is synthesized.
4. Final Cleavage:
   • After completing the sequence, the peptide is cleaved from the resin using an acidic cleavage cocktail (e.g., TFA with scavengers like TIS, EDT, and water) to remove side-chain protecting groups and release the free peptide.

Advantages of Fmoc-based SPPS

1. Mild Deprotection Conditions:
   • The use of mild basic conditions for deprotection minimizes the risk of side reactions and degradation of sensitive peptides.
2. High Yield and Purity:
   • The Fmoc strategy allows for efficient and high-yield peptide synthesis with fewer side products.
3. Automation Compatibility:
   • Fmoc-based SPPS is well-suited for automated peptide synthesizers, facilitating the rapid and consistent production of peptides.
4. Wide Range of Amino Acid Derivatives:
   • A wide range of Fmoc-protected amino acids and derivatives are commercially available, enabling the synthesis of complex and modified peptides.

Challenges and Considerations

1. Base Sensitivity:
   • Some peptides or specific amino acid residues might be sensitive to the basic conditions used for Fmoc deprotection, requiring careful optimization.
2. Handling of Side Reactions:
   • During the deprotection step, side reactions such as aspartimide formation can occur, particularly with sequences containing aspartic acid, necessitating specific protective strategies.

Green Approaches

The most widely used solvents needed for Fmoc solid-phase peptide synthesis (SPPS) are these three – N,N-Dimethylformamide (DMF), N-methyl-2-pyrrolidone, and dichloromethane (DCM). All three are hazardous chemicals but they are used primarily in washing, deprotection, and coupling steps. As a result of their toxic nature, alternatives have been sought that are less impactful on the environment or easier to dispose of in a responsible manner,.

One group discussed the use of 2-methyltetrahydrofuran (2-MeTHF) as a green solvent for coupling but using DMF for Fmoc removal and for washing (Jad et al., 2016).

In conclusion, the Fmoc strategy in SPPS provides a robust, efficient, and flexible method for synthesizing peptides, making it a preferred approach in both research and industrial applications,

References

Jad, Y. E., Acosta, G. A., Govender, T., Kruger, H. G., El-Faham, A., de la Torre, B. G., & Albericio, F. (2016). Green solid-phase peptide synthesis 2. 2-Methyltetrahydrofuran and ethyl acetate for solid-phase peptide synthesis under green conditions. ACS Sustainable Chemistry & Engineering, 4(12), pp. 6809-6814 (Article).