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Peptide Synthesis and Protecting Groups

Dec 4, 2025

Overview

  • Lecture part two on amino acids and peptides, focusing on protecting groups and solution-phase peptide synthesis.
  • Covers methods to remove common N- and C-protecting groups, methyl/benzyl ester cleavage, DCC coupling, and two directions for solution-phase synthesis (N-to-C and C-to-N).
  • Includes worked example: preparing a tripeptide (Phe–Ala–Gly) by both approaches.

Protecting Groups: Key Points

  • Common N-protecting groups: Boc (t‑butyloxycarbonyl), Fmoc (9‑fluorenylmethoxycarbonyl), CBZ (carbobenzyloxy).
    • Boc and CBZ are acid-sensitive: removed with HCl or TFA (trifluoroacetic acid).
    • Fmoc is base-sensitive: removed with strong organic bases (piperidine or triethylamine).
  • C-terminus protection: methyl ester (–COOMe) and benzyl (Bn) ester (–COOBn).
    • Methyl and benzyl esters can be saponified (base-promoted hydrolysis).
    • Benzyl esters can also be removed by hydrogenation (H2, Pd or Pt catalyst) to yield the carboxylic acid.
  • Mechanistic notes (exam relevance):
    • DCC coupling mechanism and suppression of racemization is important.
    • Some deprotection mechanisms (e.g., detailed Boc/CBZ cleavage steps) may be asked; deep mechanistic detail of every deprotection is not required for upcoming exam.

Deprotection Methods Summary (with conditions)

| Protecting Group / Functional | Typical Reagent or Condition | Result | | Boc (N‑protecting) | HCl or TFA (strong acid) | Cleaves Boc → free NH2 | | CBZ (N‑protecting) | HCl or TFA; or hydrogenation (H2, Pd/Pt) | Cleaves CBZ → free NH2 | | Fmoc (N‑protecting) | Piperidine or triethylamine (base) | Cleaves Fmoc → free NH2 | | Methyl ester (C‑protecting) | Base saponification (NaOH), then acidify (H+) | Ester → carboxylate (Na+), then carboxylic acid | | Benzyl ester (C‑protecting) | Saponification or hydrogenation (H2, Pt/C) | Benzyl ester → carboxylic acid |

Solution-Phase Peptide Synthesis: General Concepts

  • Performed in solution (commonly DMF, an aprotic solvent).
  • Two main synthetic directions:
    1. Start from the N-terminal (internal) end and extend toward C-terminal (rightward on instructor slides).
    2. Start from the C-terminal end and extend toward N-terminal.
  • Basic cycle when adding one amino acid (N‑terminal approach example):
    1. Use an N‑protected amino acid with a free carboxyl group.
    2. Couple with an incoming amino acid that has a protected C‑terminus (ester) and a free NH2 (or vice versa for C‑terminal approach).
    3. Use DCC to activate carboxyl and promote amide (peptide) bond formation (condensation; H2O lost).
    4. Hydrolyze the methyl/benzyl ester (saponify) and acidify to free the carboxyl for next coupling.
    5. Repeat steps to elongate chain.
  • Final global deprotection: remove N- and C-protecting groups (conditions depend on protecting groups used).

Role and Use of DCC

  • DCC (dicyclohexylcarbodiimide) structure: two cyclohexyl groups attached to a carbodiimide (-N=C=N-).
  • Functions:
    • Activates carboxylic acid for amide bond formation (peptide bond).
    • Minimizes racemization at the α‑carbon during coupling (critical for preserving stereochemistry).
  • Reaction outcome: amide bond forms; water is lost (condensation).

Typical Coupling Cycle (stepwise)

  • Starting material example (N‑terminal approach):
    • N‑protected amino acid (urethane form; e.g., Boc or CBZ) with free COOH.
    • Incoming amino acid: C‑protected as methyl ester, free NH2.
  • Steps:
    1. Couple with DCC → form peptide bond.
    2. Saponify methyl ester (NaOH) → form carboxylate (Na+).
    3. Acidify (H+) → regenerate COOH for next coupling.
    4. Repeat cycle for each residue.
  • Final: treat with HCl or TFA to remove Boc/CBZ (or base for Fmoc), yielding fully deprotected peptide.

Worked Example: Making Phe–Ala–Gly (tri‑peptide) — N‑terminal Approach

  • Target peptide (N→C): Phe–Ala–Gly, with N‑terminus free NH2 and C‑terminus free COOH.
  • Steps (internal/N→C build):
    1. Start with N‑protected Phe (Boc/CBZ/Fmoc) with free COOH.
    2. Add Ala–OMe (methyl ester) with free NH2; couple with DCC → form Phe–Ala(OMe).
    3. Saponify methyl ester (NaOH), then acidify → Phe–Ala–COOH.
    4. Add Gly–OMe (or other C‑protected Gly) with free NH2; couple with DCC → form protected tripeptide.
    5. Deprotect C‑terminus by saponification (or hydrogenation if benzyl).
    6. Remove N‑protecting group (HCl/TFA for Boc or CBZ; piperidine/triethylamine for Fmoc).
    7. Result: Phe–Ala–Gly, fully deprotected.

Worked Example: Making Same Tripeptide — C‑terminal Approach

  • Start with Gly–OMe (C‑protected) with free NH2.
  • Bring in N‑protected Ala (Boc/CBZ/Fmoc) with free COOH; couple with DCC → Gly–Ala(OMe).
  • Deprotect the growing chain’s N-terminus as required (HCl/TFA for Boc/CBZ; base for Fmoc).
  • Add N‑protected Phe (with COOH free) to extend to Gly–Ala–Phe (depending on drawing orientation).
  • After final coupling, remove protecting groups (saponify esters, deprotect N groups) to yield full free peptide.
  • Important orientation note: drawing/building orientation can be rotated 180° relative to naming convention; always be careful to identify N-terminus (NH2) and C-terminus (COOH) when naming/writing sequences.

Naming and Drawing Conventions (Important Clarification)

  • Standard convention: write peptide sequences from N‑terminus (left) → C‑terminus (right).
    • Example: Phe–Ala–Gly means Phe is N‑terminal residue, Gly is C‑terminal residue.
  • A structure can be drawn flipped (rotated 180°) without changing the molecule, but the written sequence must follow N→C order.
  • Changing the order of three-letter codes (e.g., Gly–Ala–Phe vs Phe–Ala–Gly) represents different peptides, not just rotated drawings.
  • Always indicate which end is NH2 (N‑terminus) and which is CO2H (C‑terminus) to avoid ambiguity.

Key Terms and Definitions

  • Peptide bond (amide bond): linkage between carboxyl carbon and amide nitrogen formed by condensation (loss of H2O).
  • Saponification: base-promoted hydrolysis of an ester to give carboxylate salt and alcohol.
  • Hydrogenation: H2 addition in presence of Pd/Pt catalyst; used to remove benzyl-type protecting groups.
  • Racemization: conversion of a stereocenter to a racemic mixture; undesirable during peptide coupling.
  • Urethane (carbamate): functional group present when N is protected (e.g., Boc is a carbamate/urethane-like group).

Action Items / Next Steps

  • Review pages 5–6 of the peptides handout: removal of common N-protecting groups (page 5) and methyl/benzyl ester removal (page 6).
  • Practice a few coupling cycles on paper for both N→C and C→N solution-phase approaches.
  • Practice naming sequences N→C and verifying orientation of drawn structures (flip drawings and confirm naming).
  • Prepare to know:
    • Reagents and conditions for removing Boc, CBZ, Fmoc, methyl esters, benzyl esters.
    • Role and structure of DCC and why it minimizes racemization.
    • Basic steps for one coupling cycle and for final global deprotection.

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