Industry Innovation Toward TFA-Free Peptide Synthesis

Solid-phase peptide synthesis (SPPS) has long relied on trifluoroacetic acid (TFA) as the standard reagent for global side-chain deprotection and resin cleavage due to its high efficiency in breaking protecting group linkages.

However, increasing concerns surrounding environmental impact, chemical stability, and process limitations have accelerated the search for alternative strategies.

TFA is classified as a persistent chemical substance and is increasingly associated with regulatory pressure, including proposed EU restrictions on per- and polyfluoroalkyl substances (PFAS), which may include TFA in future frameworks.

Key limitations of TFA-based SPPS include:

• Non-recyclable solvent burden and environmental sustainability concerns
• Dependence on acid-sensitive protecting groups
• Aggregation issues caused by hydrophobic protecting groups (e.g., tBu, Boc, Trt)
• Degradation of sensitive sequences such as N-methylated peptides under strong acidic conditions
• Prolonged deprotection requirements for groups such as Pbf-Arg, increasing risk of peptide backbone cleavage

These challenges have driven the development of next-generation orthogonal protecting group strategies for peptide synthesis under milder and more sustainable conditions.

A New Fmoc/Pic Photocatalytic SPPS Platform

A recent study published by Professor Ping Wang’s group at Shanghai Jiao Tong University (JACS) introduces a novel Fmoc/Pic (pyridylmethyl) protecting group strategy that enables:

• Orthogonal side-chain protection
• Visible-light-driven deprotection via photocatalytic C–heteroatom bond cleavage
• Acid-free global deprotection
• Full compatibility with automated peptide synthesizers

This system replaces traditional acid-labile chemistry with a photoredox catalytic platform, offering a sustainable alternative to TFA-based peptide synthesis.

Photocatalytic Deprotection via Visible Light Chemistry

The method relies on photoredox catalysis to achieve efficient cleavage of C–heteroatom bonds in amino acid side-chain protecting groups.

Using Fmoc/Pic-protected serine as a model substrate, optimized conditions were identified:

• Light source: 10 W compact fluorescent lamp (CFL)
• Catalyst: Ru(bpy)₃Cl₂ (1.5 mol%)
• Reducing agent: Ascorbic acid (5.0 equiv.)
• Solvent system: PBS/MeOH (pH 5.0)

Under these conditions, complete deprotection was achieved within 20 minutes with quantitative conversion.

Key mechanistic insights include:

• Optimal reactivity observed at pH 4.0–5.0, indicating the importance of pyridinium protonation
• Blue or green LED irradiation reduced efficiency due to suboptimal absorption alignment with Ru(bpy)₃²⁺
• Alternative photocatalysts such as Eosin Y showed insufficient redox potential
• Organic photocatalyst 4-CzIPN demonstrated comparable efficiency, confirming metal-free feasibility

Control experiments confirmed that light, photocatalyst, and reductant are all essential for the transformation.

Broad Amino Acid Scope and Functional Group Compatibility

The Fmoc/Pic platform was successfully extended to a wide range of amino acids with tailored protecting group designs:

• Aspartic acid & Glutamic acid: Dmpic protection prevents cyclization and pyroglutamate formation
• Arginine, Lysine, Tryptophan, Histidine: Modified Pic derivatives reduce undesired side reactions
• Compatible with phospho-amino acids and non-natural amino acids

Importantly, the system enables selective cleavage of C–O, C–N, and C–S bonds under identical mild conditions, while maintaining full compatibility with:

• Boc
• Benzyl
• Phenolic protecting groups
• Ester functionalities

This represents a significant improvement over traditional acid-mediated deprotection chemistry.

Comparison with Conventional TFA-Based SPPS

Compared to standard TFA-mediated deprotection strategies, the Fmoc/Pic system offers:

• Mild aqueous reaction conditions
• No generation of reactive tert-butyl cations
• Elimination of side reactions such as:
  • Sulfonium formation
  • Irreversible S-alkylation
  • Aromatic side-chain modification

Notably, Arg(Pbf) deprotection—one of the most challenging steps in SPPS—is achieved rapidly and cleanly under photochemical conditions without backbone degradation.

This provides a significant advantage for peptides containing sensitive residues such as cysteine, tyrosine, and tryptophan.

Resin Compatibility and Fully Automated Photocleavage SPPS

To fully eliminate TFA usage, acid-sensitive resins such as Sieber amide resin and 2-chlorotrityl resin were employed for C-terminal peptide synthesis.

After peptide assembly, global deprotection and cleavage were achieved via visible-light irradiation.

Key achievements include:

• Efficient synthesis of 9 C-terminal amide peptides (52–65% yields for bioactive peptides such as oxytocin and terlipressin)
• Excellent stability of methionine-containing sequences without oxidation protection additives
• Successful synthesis of long and complex peptides (>40 amino acids) with multiple Pic modifications
• Improved peptide hydrophilicity and reduced HPLC retention times

Notably, challenging therapeutic peptides such as:

• Salmon calcitonin
• Pramlintide
• HIV-1 protease fragment peptides

were successfully synthesized with high efficiency and purity.

Fully Integrated Photocatalytic SPPS Automation

A novel photocleavable linker system was further developed, enabling:

• Simultaneous peptide release and global deprotection under visible light
• Integration of external light sources into automated peptide synthesizers
• Fully automated SPPS workflows without manual TFA cleavage

Using this system, biologically active peptides (28–38) were efficiently produced with excellent yields, including phosphorylated peptide analogs that are typically difficult to synthesize using conventional acid-based methods.

Industrial and Scientific Impact

The Fmoc/tBu SPPS strategy has dominated peptide synthesis for over two decades. However, its limitations in sustainability and chemical compatibility highlight the need for next-generation solutions.

The Fmoc/Pic photoredox platform demonstrates:

• A sustainable, acid-free peptide synthesis route
• Broad functional group tolerance
• Compatibility with automated manufacturing systems
• Enhanced stability for acid-sensitive therapeutic peptides