Limited time offer: Save on peptides!

Synthesis of Radiolabel peptides

12/31/20253 min read

1. Peptide Assembly (Fmoc-SPPS)

  • Resin: Rink amide–MBHA.

  • Synthesis: Standard Fmoc cycles

    • Manual coupling (large scale) or

    • Biotage Syro II automated synthesizer (small scale).

  • Complete linear peptide sequence on resin.

2. Cleavage & Crude Peptide Preparation

  • Cleavage: TFA-based cocktail.

  • Precipitate in cold isopropyl ether.

  • Centrifuge (3 min, 3000 rpm).

  • Wash solid with isopropyl ether (×2).

  • Dry under vacuum (2 h).

  • Dissolve crude peptide in MeCN/H₂O (40:60, v/v).

3. TATA Bicyclization (Solution Phase)

  • Peptide concentration: ~1 mM.

  • Add TATA scaffold (1.0 eq).

  • Adjust to pH 8.0 using 1.0 M NH₄HCO₃ (aq.).

  • Stir at RT.

  • Monitor cyclization by LC–MS.

4. Quench, Purification & Characterization

  • Quench with cysteine (10 eq vs peptide).

  • Adjust pH to 7.0 (1 M HCl).

  • Lyophilize.

  • Purify by RP-HPLC.

  • Pool fractions with correct MW and purity.

  • Confirm identity by MALDI-TOF + HPLC or LC–MS.

  • Lyophilize purified bicyclic peptide.

Radiolabeling of Peptides

A. ⁶⁸Ga Radiolabeling

  1. Buffering:

    • Mix HEPES buffer (1.0 M, pH 4.03) + ⁶⁸Ga-eluate (30–120 MBq) in equal volumes (40–150 µL).

    • Adjust pH to 4.0 if needed (10% NaOH).

  2. Labeling:

    • Add precursor: 2–5 nmol (1 mM in DMSO).

    • Heat 95 °C, 5 min.

  3. QC & Use:

    • Analyze by HPLC + iTLC.

    • Use directly if free/colloidal ⁶⁸Ga < 2%.

    • Dilute with 0.9% NaCl, adjust to pH ~7 (30% NaOH).

B. ¹⁷⁷Lu Radiolabeling

  1. Reaction Setup:

    • NH₄OAc buffer (50 µL, pH 5.4).

    • Precursor: 2–5 nmol (1 mM in DMSO).

    • Add [¹⁷⁷Lu]LuCl₃ (20–100 MBq) in 0.04 M HCl.

  2. Labeling:

    • Heat 95 °C, 15 min.

  3. QC & Use:

    • Analyze by HPLC + iTLC.

    • Use without purification.

    • Dilute with 0.9% NaCl, adjust to pH ~7.

C. ¹¹¹In Radiolabeling

  1. Reaction Setup:

    • NH₄OAc buffer (0.1 M, pH 5.5, 144 µL).

    • Precursor: 1.5 nmol (0.2 mM in DMSO).

    • Add [¹¹¹In]InCl₃ (65 MBq).

  2. Labeling:

    • Heat 90 °C, 10 min, shake 500 rpm.

  3. QC & Purification:

    • Analyze by HPLC + iTLC.

    • Purify using C18 Sep-Pak (EtOH/PBS elution).

  4. Formulation:

    • Final volume: 1 mL PBS.

    • Add 5 mg sodium ascorbate + 0.05% Tween-20.

Practical Notes

  • pH control is critical:

    • ⁶⁸Ga: pH 3.5–4.5 (optimal chelation).

    • ¹⁷⁷Lu / ¹¹¹In: pH 5.0–5.5.

  • Short heating times minimize radiolysis and peptide degradation.

  • Sodium ascorbate effectively suppresses radiolysis in ¹¹¹In products.

  • Avoid excess DMSO (>10%) to prevent reduced labeling efficiency.

Troubleshooting

  • Low radiochemical yield:

    • Check buffer pH and metal contamination.

    • Increase precursor amount slightly (up to 10 nmol).

  • High colloidal metal fraction:

    • Reduce heating time or metal activity.

    • Improve buffer purity and pH accuracy.

  • Poor in vivo stability:

    • Add antioxidants (ascorbate, methionine).

    • Minimize delay between labelling and injection.

Radiation Safety Measures

  • All radiolabelling performed in designated hot cells or shielded fume hoods.

  • Wear dosimeter, lab coat, double gloves, and eye protection.

  • Use lead shielding for ⁶⁸Ga and tungsten shielding for ¹⁷⁷Lu/¹¹¹In.

  • Monitor contamination regularly using GM counter or dose calibrator.

  • Dispose radioactive waste according to institutional and regulatory guidelines.

HPLC and Radio-HPLC Analyses

Analytical HPLC

HPLC analyses were performed using two methods:

  • Method A:
    Agilent 1100 series HPLC (Agilent Technologies) equipped with a Chromolith RP-18e column (100 × 4.6 mm, 2 µm, 130 Å; Merck).

  • Method B:
    Agilent 1290 UPLC (Agilent Technologies) equipped with an ACQUITY UPLC CSH C18 column (150 × 2.1 mm, 1.7 µm, 130 Å).

UV detection was performed at 220 nm and/or 254 nm.

Radio-HPLC

Radio-HPLC analyses for ⁶⁸Ga- and ¹⁷⁷Lu-labeled peptides were conducted on an Agilent 1100 series HPLC system equipped with a RAMONA Star radiodetector (Elysia Raytest).

For ¹¹¹In-labeled peptides, radio-HPLC was performed using an Agilent 1100 series HPLC equipped with a Jupiter C18 column (150 × 2.6 mm, 5 µm, 300 Å; Phenomenex) and a Raytest Gabi radiodetector (Elysia Raytest).

In all cases, separations were carried out using linear gradients with the following mobile phases:

  • Solvent A: 0.1% TFA in Milli-Q H₂O

  • Solvent B: 0.1% TFA, 10% Milli-Q H₂O in MeCN

Flow rates ranged from 0.4–1.0 mL/min, depending on column and method.

Radio-TLC Analyses

Radio-TLC analyses for ⁶⁸Ga- and ¹⁷⁷Lu-labeled peptides were performed on iTLC-SG glass microfiber paper (Agilent) using 0.5 M NH₄OAc(aq.):DMF (1:1, v/v) as the mobile phase.

For ¹¹¹In-labeled peptides, iTLC was developed using 0.1 mM NH₄OAc(aq.) containing 25 nM EDTA.

TLC plates were analyzed for 5 min using either a miniGITA Star (Elysia Raytest) or a Cyclone Plus phosphor imager (PerkinElmer).

Reference:

Sharma, A.K., Gupta, K., Mishra, A., Lofland, G., Chen, S.Y., Marsh, I., Fair, P.T., Hobbs, R.F., Armstrong, T.M., Jaffee, E.M., Gabrielson, E.W., Zheng, L., Nimmagadda, S. (2025). EphA2-targeted alpha-particle theranostics for enhancing PDAC treatment. Theranostics, 15(10), 4229-4246. https://doi.org/10.7150/thno.106948