[¹⁸F]-Organotrifluoroborates as Radioprosthetic Groups for PET Imaging: From Design Principles to Preclinical Applications

ConspectusPositron emission tomography (PET) is revolutionizing our ability to visualize in vivo targets for target validation and personalized medicine. Of several classes of imaging agents, peptides afford high affinity and high specificity to distinguish pathologically distinct cell types by the presence of specific molecular targets. Of various available PET isotopes, [¹⁸F]-fluoride ion is preferred because of its excellent nuclear properties and on-demand production in hospitals at Curie levels. However, the short half-life of ¹⁸F and its lack of reactivity in water continue to challenge peptide labeling. Hence, peptides are often conjugated to a metal chelator for late-stage, one-step labeling. Yet radiometals, while effective, are neither as desirable nor as available as [¹⁸F]-fluoride ion. Despite considerable past success in identifying semifeasible radiosyntheses, significant challenges continue to confound tracer development. These interrelated challenges relate to (1) isotope/prosthetic choice; (2) bioconjugation for high affinity; (3) high radiochemical yields, (4) specific activities of >1 Ci/μmol to meet FDA microdose requirements; and (5) rapid clearance and in vivo stability. These enduring challenges have been extensively highlighted, while a single-step, operationally simple, and generally applicable means of labeling a peptide with [¹⁸F]-fluoride ion in good yield and high specific activity has eluded radiochemists and nuclear medicine practitioners for decades.Radiosynthetic ease is of primordial importance since multistep labeling reactions challenge clinical tracer production. In the past decade, as we sought to meet this challenge, appreciation of reactions with aqueous fluoride led us to consider organotrifluoroborate (RBF₃^–) synthesis as a means of rapid aqueous peptide labeling. We have applied principles of mechanistic chemistry, knowledge of chemical reactivity, and synthetic chemistry to design stable RBF₃^–s. Over the past 10 years, we have developed several new [¹⁸F]-RBF₃^– radioprosthetic groups, all of which guarantee radiosynthetic ease while in most cases providing high tumor:nontumor (T:NT) ratios and moderate-to-high tumor uptake. Although others have developed methods for labeling of peptides with [¹⁸F]-silylfluorides or [¹⁸F]-Al-NOTA chelates, this Account focuses on the synthesis of [¹⁸F]-organotrifluoroborates.In this Account, I detail mechanistic, kinetic, thermodynamic, synthetic, and radiosynthetic approaches that enabled the translation of fundamental principles regarding the chemistry of RBF₃^–s into a tantalizingly close realization of a clinical application of an [¹⁸F]-organotrifluoroborate–peptide conjugate for imaging of neuroendocrine tumors and the generalization of this method for labeling of several other peptides

Base-Promoted Protodeboronation of 2,6-Disubstituted Arylboronic Acids

Facile based promoted deboronation of electron-deficient arylboronate esters was observed for arylboronates containing two ortho electron-withdrawing group (EWG) substituents. Among 30 representative boronates, only the diortho-substituted species underwent facile C–B fission in aqueous basic conditions (200 mM hydroxide). These results provide fundamental insight into deboronative mechanisms with implications for cross-coupling reactions, regioselective deuteration/tritiation for isotopic labeling, and the design of new ¹⁸F-trifluoroborate radioprosthetics

Stereoselective Synthesis of Brevianamide E

The hydroxypyrroloindolenine (Hpi) motif forms the fundamental core of the pentacyclic natural product, brevianamide E, the concise stereoselective synthesis of which, <i>via</i> oxidative cyclization, is described

Toward the Combinatorial Selection of Chemically Modified DNAzyme RNase A Mimics Active Against all-RNA Substrates

The convenient use of SELEX and related combinatorial methods of in vitro selection provides a formidable gateway for the generation of DNA enzymes, especially in the context of improving their potential as gene therapeutic agents. Here, we report on the selection of DNAzyme 12–91, a modified nucleic acid catalyst adorned with imidazole, ammonium, and guanidinium groups that provide for efficient M²⁺-independent cleavage of an all-RNA target sequence (<i>k</i>_obs = 0.06 min^–1). While Dz12–91 was selected for intramolecular cleavage of an all-RNA target, it surprisingly cleaves a target containing a lone ribocytosine unit with even greater efficiency (<i>k</i>_obs = 0.27 min^–1) than Dz9–86 (<i>k</i>_obs = 0.13 min^–1). The sequence composition of Dz12–91 bears a marked resemblance to that of Dz9–86 (<i>k</i>_obs = 0.0014 min^–1 with an all-RNA substrate) that was selected from the same library to cleave a target containing a single ribonucleotide. However, small alterations in the sequence composition have a profound impact on the substrate preference and catalytic properties. Indeed, Dz12–91 displays the highest known rate enhancement for the M²⁺-independent cleavage of all-RNA targets. Hence, Dz12–91 represents a step toward the generation of potentially therapeutically active DNAzymes and further underscores the usefulness of modified triphosphates in selection experiments

Synthesis of the Death-Cap Mushroom Toxin α‑Amanitin

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, <i>Amanita phalloides</i>. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, <i>A. phalloides</i> has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-trypta­thionine sulfoxide bridge, enantio­selective synthesis of (2<i>S</i>,3<i>R</i>,4<i>R</i>)-4,5-dihydroxy-isoleucine, and diastereo­selective sulfoxidation

Synthesis of the Death-Cap Mushroom Toxin α‑Amanitin

α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, <i>Amanita phalloides</i>. As a potent inhibitor of RNA polymerase II, α-amanitin is toxic to eukaryotic cells. Recent interest in α-amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, <i>A. phalloides</i> has been the only source of α-amanitin. Here we report a synthesis of α-amanitin, which surmounts the key challenges for installing the 6-hydroxy-trypta­thionine sulfoxide bridge, enantio­selective synthesis of (2<i>S</i>,3<i>R</i>,4<i>R</i>)-4,5-dihydroxy-isoleucine, and diastereo­selective sulfoxidation

Dual Mode Fluorescent ¹⁸F‑PET Tracers: Efficient Modular Synthesis of Rhodamine-[cRGD]₂‑[¹⁸F]-Organotrifluoroborate, Rapid, and High Yielding One-Step ¹⁸F‑Labeling at High Specific Activity, and Correlated <i>in Vivo</i> PET Imaging and <i>ex Vivo</i> Fluorescence

The design of dual mode fluorescent-PET peptidic tracers that can be labeled with [¹⁸F]­fluoride at high specific activity and high yield has been challenged by the short half-life of ¹⁸F and its aqueous indolence toward nucleophilic displacement, that often necessitates multistep reactions that start with punctiliously dry conditions. Here we present a modular approach to constructing a fluorescent dimeric peptide with a pendant radioprosthesis that is labeled in water with [¹⁸F]­fluoride ion in a single, user-friendly step. The modular approach starts with grafting a new zwitterionic organotrifluoroborate radioprosthesis onto a pentaerythritol core with three pendent alkynes that enable successive grafting of a bright fluorophore (rhodamine) followed by two peptides (cylcoRGD). The construct is labeled with [¹⁸F]­fluoride via isotope exchange within 20 min in a single step at high specific activity (>3 Ci/μmol) and in good yield to provide 275 mCi and high radiochemical purity. Neither drying of the [¹⁸F]­fluoride ion solution nor HPLC purification of the labeled tracer is required. Facile chemical synthesis of this dual mode tracer along with a user-friendly one-step radiolabeling method affords very high specific activity. In vivo PET images of the dual mode tracer are acquired at both high and low specific activities. At very high specific activity, i.e., 3.5 Ci/μmol, tumor uptake is relatively high (5.5%ID/g), yet the associated mass is below the limits of fluorescent detection. At low specific activity, i.e., 0.01 Ci/μmol, tumor uptake in the PET image is reduced by approximately 50% (2.9%ID/g), but the greater associated mass enables fluorescence detection in the tumor. These data highlight a facile production of a dual mode fluorescent-PET tracer which is validated with <i>in vivo</i> and <i>ex vivo</i> images. These data also define critical limitations for the use of dual mode tracers in small animals

Melanoma Imaging Using ¹⁸F‑Labeled α‑Melanocyte-Stimulating Hormone Derivatives with Positron Emission Tomography

Melanocortin 1 receptor (MC1R) is specifically expressed in the majority of melanomas, a leading cause of death related to skin cancers. Accurate staging and early detection is crucial in managing melanoma. Based on the α-melanocyte-stimulating hormone (αMSH) sequence, MC1R-targeted peptides have been studied for melanoma imaging, predominately for use with single-photon emission computed tomography, with few attempts made for positron emission tomography (PET). ¹⁸F is a commonly used PET isotope due to readily available cyclotron production, pure positron emission, and a favorable half-life (109.8 min). In this study, we aim to design and evaluate αMSH derivatives that enable radiolabeling with ¹⁸F for PET imaging of melanoma. We synthesized three imaging probes based on the structure of Nle⁴-cyclo­[Asp⁵-His-d-Phe⁷-Arg-Trp-Lys¹⁰]-NH₂ (Nle-CycMSH_hex), with a Pip linker (CCZ01064), an Acp linker (CCZ01070), or an Aoc linker (CCZ01071). ¹⁸F labeling was enabled by an ammoniomethyl-trifluoroborate (AmBF₃) moiety. <i>In vitro</i> competition binding assays showed subnanomolar inhibition constant (<i>K</i>_i) values for all three peptides. The ¹⁸F radiolabeling was performed via a one-step ¹⁸F–¹⁹F isotope exchange reaction that resulted in high radiochemical purity (>95%) and good molar activity (specific activity) ranging from 40.7 to 66.6 MBq/nmol. All three ¹⁸F-labeled peptides produced excellent tumor visualization with PET imaging in C57BL/6J mice bearing B16-F10 tumors. The tumor uptake was 7.80 ± 1.77, 5.27 ± 2.38, and 5.46 ± 2.64% injected dose per gram of tissue (%ID/g) for [¹⁸F]­CCZ01064, [¹⁸F]­CCZ01070, and [¹⁸F]­CCZ01071 at 1 h post-injection (p.i.), respectively. Minimal background activity was observed except for kidneys at 4.99 ± 0.20, 4.42 ± 0.54, and 13.55 ± 2.84%ID/g, respectively. The best candidate [¹⁸F]­CCZ01064 was further evaluated at 2 h p.i., which showed increased tumor uptake at 11.96 ± 2.31%ID/g and further reduced normal tissue uptake. Moreover, a blocking study was performed for CCZ01064 at 1 h p.i., where tumor uptake was significantly reduced to 1.97 ± 0.60%ID/g, suggesting the tumor uptake was receptor mediated. In conclusion, [¹⁸F]­CCZ01064 showed high tumor uptake, low normal tissue uptake, and fast clearance and is therefore a suitable and promising candidate for PET imaging of melanoma