**3.5 Radiolabeling with 99mTc**

Technetium-99 m is the most widely used isotope due to its ideal half-life of 6 hours, its low cost, and excellent imaging characteristics. Similar to 68Ga, 99mTc can be obtained from a generator (99Mo/99mTc, with a half-life of 66 hours for 99Mo) which is easily shipped, allowing worldwide use. Since the 1960s, 99mTc has been used in a variety of applications, including cancer research and cardiac assessment. 99mTc is a nearly pure gamma-emitter (88%), with the remaining 12% yielding internal conversion electrons. The application is therefore restricted to SPECT imaging (although research into therapeutic applications is also performed).

Possibilities for coupling of 99mTc to small organic compounds, peptides or proteins are nearly unlimited, in part due to the many oxidation states that 99mTc can have, ranging from +I to +VII. The isotope is obtained from the generator in a pH-neutral and isotonic saline solution, ensuring computability with nearly any peptide or protein.

One solution to radiolabel proteins is, instead of chemically modifying the protein, synthesizing it with an additional hexa-histidine chain at the *N*-terminus (His-tag). This addition mostly does not interfere with recognition sites and allows for site-specific labeling of 99mTc (in the form of 99mTc(CO)3) [64]. This is a twostep reaction, where 99mTcO4- (Technetium pertechnetate, as it is eluted from the generator) is first converted to 99mTc(CO)3 (thereby changing the oxidation state from +VII to +I, under relatively harsh conditions) and subsequently coupling 99mTc(CO)3 to the His-tag.

An alternative for 99mTc-labeling of peptides and proteins is conjugating them with the bifunctional chelator HYNIC (6-hydrazinonicotinic acid). HYNIC has been introduced to radiolabel an IgG antibody with 99mTc for infection imaging [65]. HYNIC is an established and appropriate BCA for 99mTc-labeling, because it allows rapid and efficient labeling of proteins. In addition, 99mTc-labeled HYNICconjugates can be produced with high specific activities. However, conjugation of HYNIC to a peptide of protein can be complex as the hydrazine group of HYNIC is highly nucleophilic and, when unprotected, undergoes unwanted side reactions with electrophiles. Protecting the hydrazine group of HYNIC-conjugated compounds with for example Fmoc, Cbz, and Boc, has been investigated by several research groups and is still subject of current research [66–71].

Since the HYNIC group can only coordinate to a metal through 2 donor groups at most, it is unable to saturate the technetium coordination sphere. To complete

the coordination sphere, an additional coligand, such as EDDA (ethylenediamine diacetic acid), tricine (*N*-[Tris(hydroxymethyl)-methyl]glycine), or nicotinic acid is required. However, the choice of the coligand has significant influence on the pharmacokinetics, and thus, biologic properties of the radiotracer [72]. Because the chemistry of HYNIC conjugation to a peptide or protein remains a challenge and the coordination mode of the 99mTc-HYNIC complex is still undefined, it is not possible to give a standard HYNIC-conjugation and 99mTc-radiolabeling protocol. For a review about 99mTc-HYNIC coordination chemistry see Meszaros *et al*. and references herein [73].
