An amine-derivatized, DOTA-loaded polymeric support for Fmoc Solid Phase Peptide Synthesis

Abstract

An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional Solid Phase Peptide Synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was used to synthesize a peptide-DOTA conjugate that was demonstrated to be a PARACEST MRI contrast agent. Therefore, this synthesis methodology can facilitate Fmoc SPPS of molecular imaging contrast agents.

Figure 1

NMR spectra of Hα in compounds 3∼6. The peak from Hα is marked with an arrow.

Figure 2

Detection of urokinase Plasminogen Activator (uPA) with 8. A) The proposed mechanism of uPA cleavage of 8 and the PARACEST spectra of 8 shows that an amide proton (red) is converted to an amine (blue) after uPA cleaves the ZGGR peptide ligand from the agent. B) The CEST spectrum showed a CEST effect from the amide at -52 ppm before uPA was added (red). The disappearance of this CEST effect after uPA was added (blue) was used to detect uPA. An enzyme-unresponsive agent, Yb-DOTA-Gly, shows a CEST effect at -16 ppm before and after uPA was added, which served as an internal control.

Scheme 1

Synthesis of α-amino-DOTA and a peptide-DOTA PARACEST MRI contrast agent. (a) tert-butylbromoacetate, K₂CO₃ (6 eq.), acetonitrile, 70°C, 6 hrs, 83%; (b) CBZ-Gly(Br)-OMe, K₂CO₃ (6eq.), acetonitrile, 70 °C, 6 hrs, 83%; (c) H₂, 10-Pd/C, ethanol; 2 hrs, quantitative; (d) Fmoc-Cl, zinc dust, acetonitrile, 87%; (e) 50% TFA in CH₂Cl₂, quantitative; (f) (i) Wang resin, HBTU (4 eq.), HOBt (1 eq.), DIEA (10 eq.), NMP, rt, overnight; (ii) 50% tert-BuOH in CH₂Cl₂, 70%; (g) (i) 20% piperidine, NMP; (ii) SPPS of Z-Gly-Gly-Arg(pbf) following standard Fmoc-chemistry (iii) 95% TFA, CH₂Cl₂, 4 hrs; (iv) Tm-triflate, acetonitrile, 50 °C, overnight.