Synthesis, characterization, and labeling with Tc-99m/Re-188 of peptide conjugates containing a dithia-bisphosphine chelating agent

Radiolabeling of small receptor-avid peptides at specific predetermined che lation sites with radioactive metals has been an effective approach for pro duction of target-specific radiopharmaceuticals for diagnosis and therapy o f diseases. Among various electron-donating groups found on chelator framew orks, phosphines are unique because they display versatile coordination che mistry with a wide range of transition metals. We have recently reported th e utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to f orm Tc-99m-labeled receptor-avid peptides by the preconjugation approach. H ere we report a novel strategy for labeling small peptides with both Tc-99m and Re-188 using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)pr opylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approac h. The first step in this approach involves the coupling of the correspondi ng (PH2)(2)S-2-COOH intermediate to the N-terminus of the peptide(s). Formy lation of P-H bonds with aqueous formaldehyde in the presence of HCl in eth anol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjug ates in the form of an oxidatively stable phosphonium salt. The P2S2-peptid e conjugates are generated (where the PH2 groups are converted to P(CH2OH)( 2) groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M so dium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with T c-99m is achieved by direct reduction with Sn(II) tartarate to yield the Tc -99m-P2S2-peptide conjugate in near quantitative yields. Complexation of th e BFCA conjugates with Re-188 is achieved by transchelation with Re-188 cit rate in yields of greater than or equal to 90%. in this study, (PH2)(2)S-2- COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)(2)S-2-COOH BFCA conjugate was converted to the hydroxy me thylene phosphine form and complexed with 99mTc to produce the (TcO2)-Tc-99 m-P2S2-GlyGlyOEt conjugate 8 in RCPs of greater than or equal to 95%. This singular Tc-99m product is stable over 24 h in aqueous solution as confirme d by HPLC. Identical retention times of the (TcO2)-Tc-99m-P2S2-GlyGlyOEt co mplex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The uti lity of this postconjugation strategy was further demonstrated by synthesiz ing a P2S2-D-Lys(6)-LHRH conjugate and producing its corresponding Tc-99m c omplex in RCPs of greater than or equal to 88%. Finally, the P2S2-5-AVa-BBN [7-14] NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converte d to P(CH2OH)(2) groups and subsequently labeled with Re-188 to yield a Re- 188-labeled bombesin analogue with a RCP of greater than or equal to 90%. T he biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)(2)S-2-COOH BFC A can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (Tc-99m) and therapeutic (Re-188) radionuclides vi a the postconjugation approach in high yields.