Ribosomal Translation

Solid-Phase Peptide Synthesis

Summary

Solid-phase peptide synthesis (SPPS) is a chemical method for constructing peptides by sequentially adding protected amino acids to a growing chain anchored to an insoluble resin. Developed by Bruce Merrifield, this revolutionary technique enables rapid synthesis of custom peptides and small proteins.

Key Points

  • 1Peptide built on insoluble resin support C-to-N terminus
  • 2Fmoc/tBu strategy is modern standard (base-labile α-protection)
  • 3Cycle: deprotect → wash → couple → (cap) → repeat
  • 4Final TFA cleavage releases peptide with all protections removed

Solid-phase peptide synthesis revolutionized peptide chemistry by enabling efficient, automated production of peptides for research and therapeutics.

Historical Context

The Merrifield Revolution

In 1963, R. Bruce Merrifield introduced solid-phase synthesis:

  • Anchored the growing peptide to an insoluble polymer support
  • Enabled excess reagents and simple washing to drive reactions to completion
  • Eliminated tedious purification after each coupling step

    • - Awarded the 1984 Nobel Prize in Chemistry

    Impact on Science

    SPPS enabled:

  • Routine synthesis of peptides up to ~50 residues
  • Synthesis of proteins via fragment condensation
  • Production of peptide drugs, vaccines, and research tools

    • Fundamental Principles

    The Solid Support

    The peptide is built on an insoluble resin:

    - Polystyrene-based: Cross-linked with divinylbenzene

    - PEG-based: Polyethylene glycol grafted resins (better swelling)

    - Linker: Connects first amino acid to resin, determines cleavage conditions

    C-to-N Synthesis

    Unlike ribosomal synthesis (N-to-C), SPPS proceeds C-terminus to N-terminus:

  • First amino acid attached via C-terminus to resin
  • N-terminus protected
  • Each cycle: deprotect N-terminus, couple next amino acid
  • Chain grows from C to N

    • Protection Strategy

    Amino acids require temporary and permanent protecting groups:

    - α-Amino protection: Removed each cycle (Fmoc or Boc)

    - Side chain protection: Removed at final cleavage

    - Orthogonal protection: Different conditions for different groups

    Major Strategies

    Boc/Benzyl Strategy

    The original Merrifield approach:

    - Boc (tert-butyloxycarbonyl): α-amino protection

    - Removed with TFA (trifluoroacetic acid) each cycle

  • Side chains protected with benzyl-based groups

    • - Final cleavage: HF (hydrogen fluoride) - corrosive, requires special equipment

  • Advantages: Very stable; good for difficult sequences

    • Fmoc/tBu Strategy

    The modern standard:

    - Fmoc (9-fluorenylmethyloxycarbonyl): α-amino protection

    - Removed with piperidine (base) each cycle

  • Side chains protected with tert-butyl groups

    • - Final cleavage: TFA cocktail - safer than HF

  • Advantages: Milder conditions, easier automation, UV monitoring

    • The Synthesis Cycle

    Step 1: Deprotection

    Remove α-amino protecting group:

  • Fmoc: 20% piperidine in DMF (5-20 min)
  • Boc: 25-50% TFA in DCM (1-2 min)

    • Step 2: Washing

    Remove excess reagents and byproducts:

  • Multiple washes with DMF and DCM
  • Critical for preventing side reactions

    • Step 3: Coupling

    Form new peptide bond:

    - Coupling reagents: Activate carboxyl group

  • Common activators: HBTU, HATU, DIC/Oxyma
  • Excess amino acid (2-5 equivalents)
  • 15-60 minutes depending on sequence

    • Step 4: Capping (Optional)

    Acetylate unreacted amines:

  • Acetic anhydride treatment
  • Prevents deletion sequences

    • Challenges and Solutions

    Difficult Sequences

    Some sequences aggregate on resin:

  • β-sheet formation
  • Hydrophobic stretches
  • Solutions: Pseudoprolines, backbone protection, microwave heating

    • Racemization

    Risk of stereochemical scrambling:

  • Particularly for His, Cys
  • Minimized by coupling reagent choice
  • Avoided by preventing prolonged activation

    • Aspartimide Formation

    Asp residues can cyclize:

  • Forms β-aspartyl peptides
  • Prevented by backbone protection or Hmb protection

    • Cleavage and Deprotection

    TFA Cleavage Cocktails

    Fmoc synthesis uses TFA-based cleavage:

    - TFA: Removes side chain protections and cleaves linker

    - Scavengers: Trap reactive carbocations

    - Water, triisopropylsilane (TIS), ethanedithiol (EDT)

    - Specific cocktails for different side chains

    Work-up

    After cleavage:

  • Precipitate peptide in cold ether

    • - Purify by RP-HPLC

    - Characterize by mass spectrometry

    Applications

    Therapeutic Peptides

    Many drugs synthesized by SPPS:

  • Insulin analogs
  • GLP-1 agonists (semaglutide)
  • Cyclic peptides

    • Research Tools

  • Enzyme substrates and inhibitors
  • Epitope mapping
  • Protein semi-synthesis
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