Protecting Group Mechanisms in Peptides

Overview

• Lecture covers mechanisms for protecting and deprotecting amino acid/peptide functional groups.
• Focus: Boc (t-butoxycarbonyl), CBZ/Fmoc (carbamate-type) protections, and cleavage of ester and common N-protecting groups.
• Emphasis: these are primarily acyl (asil) transfer reactions and familiar substitution/elimination mechanisms.

Mechanism Fundamentals

• General asil (acyl) transfer:
  • Nucleophile attacks acyl carbon (2-in, 2-out) forming a tetrahedral intermediate.
  • Proton transfers can convert a poor leaving group into a better one (e.g., protonate O or N).
  • Collapse of the tetrahedral intermediate expels the leaving group and transfers the acyl group to the nucleophile.
• Show curved arrows from electron source toward bond being made/broken; ensure arrow tails start at electron-rich sites.

Boc Protection (t-Butoxycarbonyl)

• Reagent: Boc-anhydride (tert-butyl dicarbonate); structure recommended to be memorized.
• Mechanism summary:
  • Amine nucleophile attacks carbonyl of Boc-anhydride, forming tetrahedral intermediate.
  • Proton transfer from nitrogen to leaving group oxygen makes the leaving group better.
  • Collapse of intermediate transfers Boc group to the amine; leaving group departs as a protonated species.
• Concept: Proton transfer to leaving group is productive; without it reaction reverts.

CBZ and Fmoc Protection (Carbamate-Type / Acyl Chlorides)

• Reagents: CBZ-Cl (benzyloxycarbonyl chloride) and Fmoc-Cl (9-fluorenylmethoxycarbonyl chloride) — both are acyl chlorides differing in R group.
• Mechanistic options (both acceptable in exams):
  • Path A: Formation of tetrahedral intermediate, then proton transfer to chlorine (or leaving oxygen) to make leaving group better, followed by chloride (or HCl) departure.
  • Path B: Chloride departure directly from tetrahedral intermediate (less commonly taught now).
• Preferred teaching approach: show proton transfer to the leaving group first (analogous to Boc mechanism), then loss of HCl to give the protected amide (carbamate) and HCl byproduct.
• Key point: Nitrogen must be protonated/able to transfer H to create a superior leaving group; otherwise reaction reverses.

Cleavage of Ester Protecting Groups (Methyl/Benzyl Esters)

• Mechanism: Saponification (base-catalyzed ester hydrolysis)
  • Hydroxide attacks the ester carbonyl to form a tetrahedral intermediate.
  • Alkoxide departs, then alkoxide deprotonates the formed carboxylic acid to give carboxylate and alcohol.
• This is standard base-promoted ester hydrolysis taught previously.

Fmoc Deprotection (Base-Induced)

• Typical bases: tertiary amines (e.g., triethylamine) or stronger organic bases (e.g., piperidine).
• Mechanism summary:
  • Base deprotonates the benzylic/α-proton adjacent to the fluorenyl group, forming a stabilized anion (aromatic stabilization).
  • This triggers an E2-like process and fragmentation, producing CO2 and the liberated amine.
  • Intermediate anion decomposition is spontaneous due to aromatic stabilization and CO2 formation.
• Important: Fmoc removal proceeds by base-promoted elimination and decarboxylation to release amine.

Boc Deprotection (Notes / To Confirm)

• Instructor referenced a separate slide set for Boc cleavage (possibly omitted or deleted in this recording).
• Students should confirm Boc deprotection mechanism/material in class or with instructor (often acid-catalyzed cleavage producing tert-butanol and CO2).
• Action: Check peptide handout page 10 or consult instructor for missing Boc cleavage slide.

Key Terms and Definitions

• Asil (acyl) transfer: Transfer of an acyl group from one atom (leaving group) to a nucleophile.
• Tetrahedral intermediate: Four-coordinate intermediate formed when nucleophile adds to a carbonyl.
• Leaving group activation: Protonation of an otherwise poor leaving group to make it depart.
• Saponification: Base-catalyzed hydrolysis of esters to give carboxylate and alcohol.
• E2-like elimination (Fmoc): Base deprotonation leading to elimination and fragmentation with CO2 release.

Important Exam/Practice Guidance

• In mechanisms, redraw intermediates clearly — instructors prefer neat redraws.
• When showing proton transfers with arrows, place arrow tails at the electron pair moving toward the proton and show the bond-breaking arrow correctly.
• Either of the accepted mechanistic depictions for acyl chloride reactions (chloride leaving first vs. proton transfer then chloride leaving) will usually be accepted, but demonstrating proton transfer first aligns with Boc mechanism teaching.
• Memorize Boc-anhydride (tert-butyl dicarbonate) structure; many mechanisms reference it.
• Practice the set of 18 asil-transfer examples previously provided by instructor.

Action Items / Next Steps

• Review peptide handout, especially page 9 (reactions 14–15) and page 10 for Boc cleavage.
• Confirm missing Boc cleavage slide/content with the instructor (Dr. P).
• Practice drawing acyl transfer mechanisms with clear curved arrows and proton transfers.
• Revisit prior chapters on acyl transfer and saponification for mechanism reinforcement.