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An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with 99mTc at room temperature


The mercaptoacetyltriglycine (MAG3) chelator has been shown to stably complex technetium-99m (99mTc) for nuclear imaging and radiorhenium (186/188Re) for tumor radiation therapy studies. The bifunctional N-hydroxysuccinimidyl ester of MAG3 with S-acetyl protection (N-hydroxysuccinimidyl S-acetylmercaptoacetyltriglycinate (NHS-MAG3)) has been successfully used to covalently conjugate a MAG3 chelator to primary amine functionalized biomolecules. We describe herein a simplified synthesis of NHS-MAG3 that begins with the preparation of the N-hydroxysuccinimidyl ester of S-acetylmercaptoacetic acid (N-succinimidyl S-acetylmercaptoacetate (SATA)) from mercaptoacetic acid and is followed by the synthesis of S-acetylmercaptoacetyltriglycine from SATA, together requiring about 14 days. Finally, the synthesis of NHS-MAG3 from S-acetylmercaptoacetyltriglycine requires a further 5 days. We had earlier described a method for the preparation of MAG3-conjugated and 99mTc-radiolabeled biomolecules that required elevated temperatures during postconjugation purification. We now report a modified method for the preparation that is accomplished at room temperature and therefore applicable to temperature-sensitive biomolecules. The conjugation and radiolabeling of bovine serum albumin is used as an example. The conjugation and purification requires about 2–3 h and the radiolabeling and postlabeling purification requires about an additional 2 h.

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Figure 1
Figure 2: A mass spectrum of NHS-MAG3 synthesized by the method described in text.
Figure 3: Size-exclusion HPLC analyses of 99mTc-labeled MAG3-BSA before (trace A) and after (trace B) postlabeling purification on a P4 column.


  1. Hnatowich, D.J. et al. Labeling peptides with technetium-99m using a bifunctional chelator of a N-hydroxysuccinimide ester of mercaptoacetyltriglycine. J. Nucl. Med. 39, 56–64 (1998).

    CAS  Google Scholar 

  2. Liu, G. et al. Successful radiotherapy of tumor in pretargeted mice by 188Re-radiolabeled phosphorodiamidate morpholino oligomer, a synthetic DNA analogue. Clin. Cancer Res. 12, 4958–4964 (2006).

    Article  CAS  PubMed  Google Scholar 

  3. Lei, K. et al. Technetium-99m antibodies labeled with MAG3 and SHNH: an in vitro and animal in vivo comparison. Nucl. Med. Biol. 23, 917–922 (1996).

    Article  CAS  Google Scholar 

  4. Zhang, Y., Liu, N., Zhu, Z., Rusckowski, M. & Hnatowich, D.J. Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to antisense DNA. Eur. J. Nucl. Med. 27, 1700–1707 (2000).

    Article  CAS  Google Scholar 

  5. Vanderheyden, J.L. et al. Evaluation of 99mTc-MAG3-annexin V: influence of the chelate on in vitro and in vivo properties in mice. Nucl. Med. Biol. 33, 135–144 (2006).

    Article  CAS  Google Scholar 

  6. Hjelstuen, O.K., Tonnesen, H.H., Bremer, P.O. & Verbruggen, A.M. 3′-99mTc-labeling and biodistribution of a CAPL antisense oligodeoxynucleotide. Nucl. Med. Biol. 25, 651–657 (1998).

    Article  CAS  Google Scholar 

  7. Fritzberg, A.R., Kasina, S., Eshima, D. & Johnson, D.L. Synthesis and biological evaluation of technetium-99m MAG3 as a hippuran replacement. J. Nucl. Med. 27, 111–116 (1986).

    CAS  Google Scholar 

  8. van Gog, F.B., Visser, G.W., Gowrising, R.W., Snow, G.B. & van Dongen, G.A. Synthesis and evaluation of 99mTc/99Tc-MAG3-Biotin conjugates for antibody pretargeting strategies. Nucl. Med. Biol. 25, 611–619 (1998).

    Article  CAS  Google Scholar 

  9. Okarvi, S.M. & Jammaz, I.A. Preparation and in vitro and in vivo evaluation of technetium-99m-labeled folate and methotrexate conjugates as tumor imaging agents. Cancer Biother. Radiopharm. 21, 49–60 (2006).

    Article  CAS  Google Scholar 

  10. Rusckowski, M., Gupta, S., Liu, G., Dou, S. & Hnatowich, D.J. Investigations of a Tc-99m-labeled bacteriophage as a potential infection-specific imaging agent. J. Nucl. Med. 45, 1201–1208 (2004).

    CAS  Google Scholar 

  11. Duncan, R.J.S., Weston, P.D. & Wrigglesworth, R. A new reagent which may be used to introduce sulfhydryl-groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132, 68–73 (1983).

    Article  CAS  Google Scholar 

  12. Decristoforo, C. & Mather, S.J. Preparation, 99mTc-labeling, and in vitro characterization of HYNIC and N3S modified RC-160 and [Tyr3]octreotide. Bioconjugate Chem. 10, 431–438 (1999).

    Article  CAS  Google Scholar 

  13. Wang, Y., Liu, G. & Hnatowich, D.J. Methods for MAG3 conjugation and 99mTc radiolabeling of biomolecules. Nat. Protocols 1, 1477–1480 (2006).

    Article  CAS  Google Scholar 

  14. Winnard, P. Jr., Chang, F., Rusckowski, M., Mardirossian, G. & Hnatowich, D.J. Preparation and use of NHS-MAG3 for technetium-99m labeling of DNA. Nucl. Med. Biol. 24, 425–432 (1997).

    Article  CAS  Google Scholar 

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Correspondence to Yi Wang or Donald J Hnatowich.

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Wang, Y., Liu, X. & Hnatowich, D. An improved synthesis of NHS-MAG3 for conjugation and radiolabeling of biomolecules with 99mTc at room temperature. Nat Protoc 2, 972–978 (2007).

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