NHS–MAS3: a bifunctional chelator alternative to NHS–MAG3

This laboratory uses an N-hydroxysuccinimide derivative of S-acetylmercaptoacetyltriglycine (NHS–MAG3) to conjugate amines for subsequent labeling with 99mTc. However, the synthesis from triglycerine is general and not restricted to this tripeptide. We had earlier selected a small number of alternative tripeptides and synthesized the corresponding NHS derivatives. Each was then evaluated in a search for bifunctional chelators with properties superior to NHS–MAG3, such as lower serum protein binding or improved stability to cysteine challenge. Based on these preliminary results, NHS–S-acetylmercaptoacetyltriserine (NHS–MAS3) was selected for further investigation. We have now conjugated this bifunctional chelator to biocytin and to an amine-derivatized peptide nucleic acid (PNA). Both carriers were also conjugated with NHS–MAG3 under identical conditions and all were labeled with 99mTc at neutral pH and at boiling temperature while the conjugated PNAs were radiolabelled at neutral pH and at room temperature. Regardless of the chelator, reverse phase HPLC radiochromatograms of the labeled biotins and PNAs after purification showed a single peak. However, by size exclusion HPLC, the radiochromatograms always showed several peaks even after purification, but the MAS3 radiochromatograms were less complicated. For biotin and PNA both, radiolabeling via MAS3 showed improved 99mTc stability in 37°C serum and in cysteine solution. The four preparations were administered to mice implanted in one thigh with avidin beads (biotins) or complementary PNA beads (PNAs). At 5 h post-administration, no significant differences were observed in the targeting of PNA beads between the two chelators, however the target thigh/normal thigh ratio was significantly higher for MAS3–biotin compared to MAG3–biotin. We conclude that labeling biocytin and amine-derivatized PNA with NHS–MAS3 compared to NHS–MAG3 provides simpler radiochromatographic profiles, improved stability of the label in serum and cysteine solution and can improve in vivo targeting.