An innovative approach for the synthesis of dual modality peptide imaging probes based on the native chemical ligation approach

Abstract

The synthesis of a dual peptide-based imaging probe consists of two steps performed in aqueous solution under mild conditions.

A Chemical Strategy for the Preparation of Multimodified Peptide Imaging Probes

“…The AAZTA chelator can be complexed with gallium under mild conditions by treatment with 1 equiv of GaCl 3 in acidic aqueous buffer, at room temperature. 14,20 cyclo(-Orn(N δ -CO-C4-AAZTA, γ-S-succinimido-Cy5.5)-Arg-Gly-Asp-D-Phe-) was successfully complexed with Ga(III) ion by treatment with an equimolar amount of GaCl 3 in acetate buffer pH 4.6/CH 3 CN (1:1), at room temperature for 1 h (Figure S14) to perform cell-binding studies using a probe with the same chemical properties of the PET/OI imaging probe. Metal complexation procedure did not alter fluorophore spectroscopic properties, as the final Ga(III)-complexed probe featured the UV−vis spectrum of the parent cyanine 5.5 maleimide (Figure S15).…”

AAZTA-Derived Chelators for the Design of Innovative Radiopharmaceuticals with Theranostic Applications

“…Another more original linkage method based on the native chemical ligation (NCL) [48] was applied to AAZTA derivatives by Hawala et al [49]. This technique allowed the formation of an amide bond between a N-terminal cysteinyl peptide and a pentanoic acid-AAZTA derivative, preactivated as a sodium 2-mercaptoethanesulfonate thioester (Scheme 7).…”

“…Scheme 7. Native chemical ligation mechanism for N-terminal cysteinyl peptides bioconjugation with AAZTA thioester, as reported by Hawala et al[49].…”

Exploiting Protein N-Terminus for Site-Specific Bioconjugation

“…Although in this case the N-terminal cysteine could be restored by treatment with O-methylhydroxylamine [54,55], the most convenient strategy to prepare N-terminal cysteinyl proteins refers to the exposition of the N-terminal cysteine after protein expression and purification through proteolytic cleavage with Factor Xa, tobacco etch virus (TEV), and thrombin proteases [56][57][58] or using selfcleavable fusion partners such as intein [59]. The most powerful and widespread chemical approach exploited to target N-terminal cysteinyl proteins refers to the reaction with a thioester probe through NCL [33,54,[60][61][62][63][64][65] (Figure 2a). The NCL reaction proceeds through a reversible trans-thioesterification mediated by the thiol group of the cysteine, followed by a spontaneous intramolecular S→N acyl shift that translocates the probe on the alpha-amine group, affording an amide bonded product [22].…”

“…Notably, the thiol group of the N-terminal cysteine mediates NCL reaction but is restored in the free form in the final conjugated product. Therefore, such a thiol group could be exploited for pursuing second site-specific labeling using a thiol reactive probe, for instance, through the thiol-maleimide chemistry [64,66]. Conveniently, thioester probes can be obtained from commercially available NHS-ester derivatives by treatment with thiols [65,67].…”