𝗨𝗻𝗱𝗲𝗿𝘀𝘁𝗮𝗻𝗱𝗶𝗻𝗴 “𝗠+𝟭𝟲” 𝗜𝗺𝗽𝘂𝗿𝗶𝘁𝗶𝗲𝘀 𝗶𝗻 𝗣𝗲𝗽𝘁𝗶𝗱𝗲 𝗦𝘆𝗻𝘁𝗵𝗲𝘀𝗶𝘀

1) Lysine (δ-carbon hydroxylation)

• Position affected: δ-C of the side chain (ε-amino group is on δ-C).

• Mechanism: Often occurs under strong oxidizing conditions, e.g., during Fmoc deprotection (piperidine) in the presence of oxygen, or during TFA cleavage if traces of oxidants are present.

• Impact: Adds +16 Da (OH) to the Lys residue.

• Detection: LC-MS shows mass increase of +16 Da; may slightly change retention time in HPLC due to increased polarity.

• Preventive measures:

  • Use fresh, degassed solvents.

  • Minimize air exposure during cleavage and handling.

  • Include antioxidants (e.g., TIPS during TFA cleavage).

2) Tryptophan (β-carbon hydroxylation)

• Position affected: β-C of indole ring.

• Mechanism: Indole is highly sensitive to oxidation. Hydroxylation usually occurs during light exposure, oxygen, or strong oxidants.

• Impact: +16 Da; can lead to trp side-chain modifications like oxindole formation.

• Detection: LC-MS; UV absorbance of Trp changes due to indole modification.

• Preventive measures:

  • Protect from light.

  • Use mild cleavage conditions; avoid strong acids or oxidizing reagents if possible.

  • Consider Trp protection (Boc, O-benzyl) if long exposure is needed.

3) Proline (C3 or C4 hydroxylation)

• Position affected: γ (C3) or δ (C4) carbon of pyrrolidine ring.

• Mechanism: Often enzymatic in nature in vivo, but in SPPS, can arise from strong oxidative conditions during cleavage or resin treatment, especially if side-chain protection is absent.

• Impact: +16 Da per hydroxylation.

• Detection: LC-MS; may show altered retention due to hydrogen bonding; sometimes hard to separate.

• Preventive measures:

  • Use appropriate protecting groups (Boc or tBu on adjacent residues).

  • Avoid overexposure to oxidizing conditions during TFA cleavage.

4) Aspartate (β-carbon hydroxylation)

• Position affected: β-C (side-chain carboxyl).

• Mechanism: Usually occurs via oxidative attack under acidic or radical conditions, sometimes during activation of Asp for coupling. Racemization can accompany oxidation.

• Impact: +16 Da; may lead to Asp–Asp or Asp–X side products if β-hydroxylation alters reactivity.

• Detection: LC-MS and HPLC; may co-elute with deamidated products, careful mass spec analysis needed.

• Preventive measures:

  • Use mild coupling conditions, e.g., HBTU/HATU with minimal base excess.

  • Avoid prolonged exposure to oxygen or radical-forming reagents.

5) Methionine → Methionine Sulfoxide

• Reaction: Oxidation of the sulfur atom in the thioether side chain.

• Mass change: +16 Da per Met residue.

• Mechanism: Oxidation by oxygen, peroxides, or trace oxidants during SPPS or TFA cleavage.

• Detection: LC-MS: +16 Da; sometimes HPLC shows slight shift due to increased polarity.

• Impact: Can affect peptide activity, solubility, or folding.

• Prevention:

  • Use reducing agents (TIPS, DTT) during TFA cleavage.

  • Minimize air/oxygen exposure.

  • Store peptides at low temperature under inert atmosphere.

6) Tyrosine → 3,4-Dihydroxyphenylalanine (DOPA)

• Reaction: Hydroxylation of phenolic ring at positions 3 and 4.

• Mass change: +16 Da per hydroxylation.

• Mechanism: Radical or metal-catalyzed oxidation (Cu²⁺, Fe³⁺) during synthesis or storage.

• Detection: LC-MS +16 Da; UV absorbance shifts due to catechol formation.

• Impact: Alters polarity and reactivity; can lead to cross-linking (quinone formation).

• Prevention:

  • Avoid metal contamination in solvents/reagents.

  • Protect from light and oxygen.

  • Consider temporary protecting groups (e.g., O-benzyl) if long synthesis.

7) Histidine → Oxohistidine

• Reaction: Oxidation of the imidazole ring, forming keto-imidazole derivatives.

• Mass change: +16 Da per His oxidation.

• Mechanism: Reactive oxygen species attack imidazole, often during TFA cleavage or storage.

• Detection: LC-MS: +16 Da; may change HPLC retention.

• Impact: Can interfere with metal binding or peptide activity.

• Prevention:

  • Degassed solvents and antioxidants.

  • Minimize prolonged acid exposure or light.

8) Cysteine → Sulfenic Acid (Cys-SOH)

• Reaction: Oxidation of thiol to sulfenic acid (intermediate before sulfinic/sulfonic acids).

• Mass change: +16 Da.

• Mechanism: Air oxidation, peroxides, or during TFA cleavage if no scavenger.

• Detection: LC-MS: +16 Da; highly reactive and may form disulfides or sulfonic acids.

• Impact: Can cause disulfide scrambling or peptide aggregation.

• Prevention:

  • Use scavengers during TFA cleavage (TIPS, EDT, thioanisole).

  • Protect Cys with Acm, Trt, or tBu groups.

  • Minimize exposure to oxygen.

9) Proline → γ-Glutamyl Semialdehyde (oxidized proline)

• Reaction: Oxidation of the pyrrolidine ring to form an aldehyde at γ-C.

• Mass change: +14 Da (because of loss of two hydrogens and addition of oxygen, sometimes represented as +16 depending on tautomer).

• Mechanism: Can occur during TFA cleavage under oxidative conditions or prolonged air exposure.

• Detection: LC-MS: +14/+16 Da; retention time changes.

• Impact: Can lead to peptide backbone cross-linking (Schiff base formation).

• Prevention:

  • Minimize exposure to oxidizing conditions.

  • Include reducing scavengers during cleavage.

  • Avoid metals that catalyze oxidation.

General Notes on Hydroxylation in Peptide Synthesis

1. Hydroxylation always adds +16 Da per site, so MS analysis is straightforward.

2. More common in residues with electron-rich side chains (Trp, Lys, Pro).

3. Often occurs post-cleavage or during prolonged reaction times, not just on resin.

4. Minimizing oxygen, light, and strong oxidizing agents is the best preventive strategy.