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Jan 1, 2009 (Vol. 29, No. 1)

Tritiated Opiate Receptor Radioligands

Synthesis and Characterization: Past, Present, and Future Practices

  • Tritiation Process

    Click Image To Enlarge +
    Figure 3. [3H] Morphine

    The synthetic process to radiolabel with tritium is a multistep process. The catalyst is added to a glass flask with special sidearms and a stir bar. The appropriate precursor, a halogenated or unsaturated compound (1–50 mg), is dissolved in a minimum amount of solvent (1–2 mL), and then is also added to the flask. The flask is attached to a stainless steel vacuum line. The flask is partially evacuated, lowered into liquid nitrogen and then fully evacuated. Tritium gas is introduced to the flask to the appropriate pressure. The reaction is stirred for the designated amount of time, and pressure is monitored to track incorporation of tritium into the compound.

    Once the reaction is complete, the flask is again evacuated to remove excess tritium gas. Additional solvent is reintroduced, and the flask contents are filtered in line and transferred to a receiving flask. Labile tritium is removed by repeated additions of protic solvents (e.g., water, methanol) and repeated evacuations to dryness. The product is then dissolved in a solvent, ready for purification.

    PerkinElmer has developed an automated tritiation line to handle this process. This control improves safety, consistency, and reduces tritiated waste.

    A final tritiation technique that greatly expanded the limits of specific activity was based on the fact that many opiate-receptor compounds possess methyl groups attached to heteroatoms like nitrogen or oxygen. Exploiting this structural circumstance facilitated removal of these methyl groups from compounds, yielding N or O-desmethyl precursor analogues of them.

    As a result, methyl groups labeled at nearly theoretically high specific activity with tritium using either [3H] methyl iodide or, more recently [3H] methyl nosylate, can be reattached. Products of this methodology are compounds like [N-methyl-3H] morphine (Figure 3) and [N-methyl-3H] dihydromorphine.

    Early on, PerkinElmer observed that some of these radioligands were so highly radioactive that they manifested the curious behavior of isotopic fractionation on HPLC. Awareness of this intriguing chromatographic property was critical to their purification and characterization.

    The ongoing effort to tritium label opiate-receptor ligands has also prompted us to utilize and extend the boundaries of instrumentation to prepare and characterize these substances. Long ago, we recognized the importance and power of tritium NMR to analyze tritiated opiate ligands, and we conducted early experiments over 30 years ago. An example of this, the proton decoupled tritium NMR (CD3OD) of the semi-synthetic opiate [3H] buprenorphine, is shown in Figure 4.

    The tritium NMR spectrum of this radioligand clearly corroborates that the molecule has been labeled with tritium predominantly in the buprenorphine 15 and 16 bridgehead positions as evidenced by the large sharp multiplet peaks near 2.0 and 3.0 ppm. It also interestingly reveals that this HPLC homogeneous product contains a minor amount of nonspecific tritium incorporation in other locations of the molecule as disclosed by other smaller tritium resonances.

    Mass spectrometry has been indispensable in establishing product identity and even calculating the specific activity of highly tritiated opiates. With such small masses, highly tritiated substances cannot be accurately weighed to ascertain their mass for specific activity calculations, but mass spectrometry easily facilitates this measurement.

    The mass spectrum of high-specific activity [N-methyl-3H] morphine, clearly documents the presence of a large radioactive (M + I) parent ion at 292.5 m/z due to the incorporation of three tritium atoms in the product by tritium N-methylation (Figure 5). Comparing the size of this dominant radioactive parent peak with that of the small amount of unlabeled morphine at 286.5 m/z allows the measurement of tritiated product specific activity to be 85.5 Ci/mmol.

    Over the past several decades, significant advancement has been made in the synthesis and characterization of tritiated opiate radioligands. These days even more structurally complex and challenging opiate drug candidates are being identified. The future will demand and no doubt witness the development of newer tritiation and analytical methods to accomplish the radiolabeling of these important substances.

  • Click Image To Enlarge +
    Figure 4. Proton decoupled tritium NMR of [3H] buprenorphine
  • Click Image To Enlarge +
    Figure 5. Mass spectrum of high specific activity [N-methyl-3H] morphine


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