Proteins, astatination

This protein astatination reagent has been synthesized521,522 from an activated tin-containing ester (377) employing TBHP under mild acidic conditions to generate the electrophilic astatine (equation 233)523. 

In view of the potential therapeutic applications of At vide infra) the synthesis of stable astatinated protein molecules has attracted much effort (see Table VII). Proteins labeled with At can be prepared most reliably and unambiguously via incorporation of previously prepared pora-AtC6H4COOH by an acylation reaction with protein amino groups 53, 156, 158, 159, 178). Labeling proteins by this method was first reported by Hughes et al. 69, 71). 

All other investigations in this field were directed towards conjugating astatine with proteins. In most cases / -astatobenzoic acid (PAtBA) is produced first via diazotization PAtBA is subsequently bound to the NH2 group of protein by means of acylation with mixed anhydrides42 14. 

The most thorough investigation in this area was that of Harrison and Royle43 on astatination of rabbit immunoglobulin (IgG) which could be used in animal experiments. PAtBA was produced with > 90% radiochemical yield, and a reproducible overall yield of > 30% for labelled protein was obtained with negligible deastatination of the latter in vivo. These favourable results could be achieved most probably due to the fact that the PAtBA was prepared without an iodine carrier and that the micro amounts of the product were purified from the macro amounts of contaminates by HPLC. To bind PAtBA to the protein, acylation with mixed anhydride was used. Preparation of the mixed anhydride 7 (equation 5) could be carried out in about 20 minutes at ca 0 °C. For astatination rabbit IgG protein is dissolved in borate buffer (pH = 9.3) and then added to 7 (see equation 6) the procedure takes about 1 hour at ca 15 °C. The astatinated protein is separated from non-conjugated materials by gel filtration and eluted from the column by phosphate-buffered saline. 

While trying to elaborate optimal conditions for labelling the proteins with astatine, the authors45 8 realized that the second and the third step (equations 8 and 9) can be carried out simultaneously, thereby decreasing the synthesis time. Thus the whole procedure, including also the separation of the labelled protein (human lgG) by gel filtration, takes ca 90 minutes the overall yield for astatination was about 50%. 

An important conclusion drawn from the last two studies is that, with the help of trialkylstannyl derivatives, astatine can easily be incorporated into compounds with an aromatic ring or with an olefinic bond. This enables the method to be applicable for labelling a wide variety of biologically active species, drugs or proteins. 

Separation of astatinated proteins from compounds of lower molecular weight is carried out by gel filtration. A typical elution curve for 211At-labelled goat IgG separation is shown in Figure 4. 

In mitochondrial research the phosphate-transporting protein from rat liver mitochondria has been labeled with ° Hg-mersalyl At For protein labeling with astatine (alpha emitter) the following procedures may be u reaction of the protein with p-astatobenzoic acid < ndensation reaction with peptide bond and protein acetylation While labeling by the above procedures seems to be sufficiently stable a remarkable instability of the At-label was obserwd when astatinated protein was prepared electrophoretically 202) jjjg results of these authors indicate that the tyrosine-astatine bond is unstable. The conclusion of Vau an et al. that astatinated proteins lore as much as 50% of their biological activity and, in addition, are extremely toxic, is very serious. 

Astatine-211 is a promising radionuclide for systemic therapy1 3 due to its decay properties with a half-life of 7.2 hours and an effective emission of one a-particle per decay. However, the weakness of the astatine-protein bond formed after direct astatination1 4 has so far limited its clinical use. To overcome these problems indirect labelling methods have been tried such as the use of ALsuccinimidyl-(trialkylstannyl) benzoate as an intermediate for the astatination of antibodies using conjugation procedures.5 8... 

The aim of this paper is to study the influence of different reaction parameters on the labelling yield in the astatination of and to evaluate the merits of this anion as a possible prosthetic group for astatination of protein. 

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