Tc and solid supports

One of the drawbacks of Tc-tagged compounds is that they are typically designed in such a manner that the bioconjugates are capable of binding to the target

Scheme 18.4

receptor whether or not the radionuclide is present. In view of the fact that during labeling there is large excessive of ligand present compared to Tc (more than 10 000-fold), the unlabeled material must be removed prior to use because it can displace the Tc complex from the target site thereby reducing image quality/ accuracy. This is of course not an issue for Tc-essential compounds where the free ligand and Tc complex have completely different biodistribution profiles.

The simplest solution to this problem is to purify the labeling mixture by HPLC. This approach, although effective, is not suitable for the daily labelings done in hospitals. An alternative approach for the preparation of 'ligand-free' samples is to label compounds that are bound to an insoluble support in such a manner that upon coordination of Tc only the desired complex is released into solution. Unreacted ligand remains bound to the support and is removed by simple filtration.

There have been several approaches for carrying out solid-phase labeling with Tc and/or Re.78 80 Thornback, Ballinger and co-workers81 showed that it was possible to label an N3S chelate that was connected to a series of different solid supports through a succinimidyl linker via the ligand-thiol group. Although these systems afforded high specific activity formulations (specific activity in this instance refers to the amount of activity divided by the amount of free ligand present), radiochemical yields varied significantly (13-80%). Alternatively, the same chelate, derivatized with a peptide, was linked through the thiol to a gold support. Upon labeling with 99mTc, the desired product was isolated in excellent radiochemical yield (Scheme 18.5).82

Alberto and co-workers developed a solid-phase labeling system for the Tc(CO)^ core.83 Tridentate chelates comprised of different combinations of aliphatic and aromatic amines and carboxylates were linked to a TentaGel-type support. This resin, which is a polystyrene/poly(ethylene glycol)-based support, was selected because it swells in water and is stable to elevated reaction temperatures. Cleavage of the tertiary amine linker proceeded in modest to good yields with 99Tc and 99mTc but interestingly not with Re. For reactions involving 99mTc(CO)^, the products produced had very high specific activities (refer to the above definition).

Me Me

Scheme 18.5

Beyond labeling, solid-phase synthesis (SPS) is also starting to play an increasingly important role in the development of new radiopharmaceuticals. SPS affords the opportunity to prepare libraries of chelate-biomolecule conjugates as a more rapid and efficient means of identifying viable Tc radiopharmaceuticals. The majority of SPS research involving Tc has been focused on the synthesis of peptide conjugates.

The preparation of peptide-targeted Tc radiopharmaceuticals is well suited to SPS because most bifunctional Tc ligands contain amino or carboxylic acid linker groups. Standard automated peptide synthesis protocols can be used to link these bifunctional ligands to resin-bound peptides and to vary the factors which have the greatest impact on receptor-binding affinity and biodistribution of technetium-labeled peptides.84 These include the site of attachment of the Tc ligand, the presence and type of a spacer group between the ligand and the peptide, and the nature of the Tc-ligand complex (charge, size, lipophilicity, etc.). SPS offers a convenient means of identifying optimal combinations of these factors because libraries of bioconjugates can be prepared in parallel for those instruments that have multiple reaction wells.

Several methods have been developed for linking Tc ligands to peptides on solid supports. Our group showed that N3S-type chelates and their Re complexes could be conveniently incorporated into peptides when bound to Merri-field-type resins (Scheme 18.6).85 Hoffman etal. used a similar method for linking a chelate and its Re complex, through a variety of different spacer groups, to a peptide which targets bombesin receptors on tumor cells.86 Gariepy and co-workers described a method for adding an N2S2 chelate (a diamidodithiol ligand) to the N-terminus of peptides bound to a support.87

Blower and co-workers recently described a novel SPS method in which a HYNIC derivative of 9-fluorenylmethyl carbamate (Fmoc)-protected lysine (Figure 18.7) was used as a metal-binding amino acid analogue.88 This particular

tN NrOa

OH [NBu4][TcOCl4], Na gluconate




Scheme 18.6

Figure 18.7 HYNIC derivative of 9-fluorenylmethyl carbamate (Fmoc) protected-lysine; a metal-binding amino acid analogue ligand is attractive because it can be incorporated within the backbone of peptide, as opposed to simple N or C termini conjugation, using standard Fmoc solidphase peptide chemistry. The lysine-HYNIC ligand was used to prepare a Tc-labeled analogue of salmon calcitonin, a 32-amino acid neuropeptide, as a means of targeting the radionuclide to sites of bone disease and tumors. An analogous system which can bind the {TcO}3+ core was reported by Hunter and Luyt. They described the synthesis of an Fmoc-protected-lysine-derived bifunctional chelator that can be incorporated into growing peptides.89

Our group, as part of a collaborative research effort, recently described the development of a new SPS method for the preparation of Tc(I)- and Re(I)-labeled peptides.90 We showed that a tridentate chelate derived from lysine (referred to as a single amino acid chelate or SAAC) which binds the Tc(CO)^ core can be incorporated into the backbone of peptides using a conventional automated synthesizer (Scheme 18.7). One particular advantage to working


(1) 20% piperidine/DMF

(2) Wash cycle

(1) 20% piperidine/DMF

(2) Wash cycle



with the M(CO)^ core is that the coordination complexes are by and large inert (discussed earlier). In the case of the SAAC ligand, this feature makes it possible to incorporate the Re complex into the peptide using exactly the same procedure that is used to prepare the ligand. This affords a convenient means of preparing well-defined reference standards, for when it is time to label the peptide-ligand conjugates with 99mTc. As mentioned previously, having a fully characterized product is an essential part of developing and receiving approval for any new radiopharmaceutical.

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