David M. Perrin

Toward [18F]-labeled aryltrifluoroborate radiotracers: in vivo positron emission tomography imaging of stable aryltrifluoroborate clearance in mice.

The use of a boronic ester as a captor of aqueous [(18)F]-fluoride has been previously suggested as a means of labeling biomolecules in one step for positron emission tomography (PET) imaging. For this approach to be seriously considered, the [(18)F]-labeled trifluoroborate should be humorally stable such that it neither leaches free [(18)F]-fluoride to the bone nor accumulates therein. Herein, we have synthesized a biotinylated boronic ester that is converted to the corresponding trifluoroborate salt in the presence of aqueous [(18)F]-fluoride. In keeping with its in vitro aqueous kinetic stability at pH 7.5, the trifluoroborate appears to clear in vivo quite rapidly to the bladder as the stable trifluoroborate salt with no detectable leaching of free [(18)F]-fluoride to the bone. When this labeled biotin is preincubated with avidin, the pharmacokinetic clearance of the resulting complex is visibly altered. This work validates initial claims that boronic esters are potentially useful as readily labeled precursors to [(18)F]-PET reagents.

A self-cleaving DNA enzyme modified with amines, guanidines and imidazoles operates independently of divalent metal cations (M2+).

The selection of modified DNAzymes represents an important endeavor in expanding the chemical and catalytic properties of catalytic nucleic acids. Few examples of such exist and to date, there is no example where three different modified bases have been simultaneously incorporated for catalytic activity. Herein, dCTP, dATP and dUTP bearing, respectively, a cationic amine, an imidazole and a cationic guanidine, were enzymatically polymerized on a DNA template for the selection of a highly functionalized DNAzyme, called DNAzyme 9-86, that catalyzed (M2+)-independent self-cleavage under physiological conditions at a single ribo(cytosine)phosphodiester linkage with a rate constant of (0.134 ± 0.026) min−1. A pH rate profile analysis revealed pKas of 7.4 and 8.1, consistent with both general acid and base catalysis. The presence of guanidinium cations permits cleavage at significantly higher temperatures than previously observed for DNAzymes with only amines and imidazoles. Qualitatively, DNAzyme 9-86 presents an unprecedented ensemble of synthetic functionalities while quantitatively it expresses one of the highest reported values for any self-cleaving nucleic acid when investigated under M2+-free conditions at 37°C.

Substituent Effects on Aryltrifluoroborate Solvolysis in Water: Implications for Suzuki−Miyaura Coupling and the Design of Stable 18F-Labeled Aryltrifluoroborates for Use in PET Imaging

Whereas electron withdrawing substituents retard the rate of aryltrifluoroborate solvolysis, electron-donating groups enhance it. Herein is presented a Hammett analysis of the solvolytic lability of aryltrifluoroborates where log(k(solv)) values correlate to sigma values with a rho value of approximately -1. This work provides a predictable rubric for tuning the reactivity of boron for several uses including (18)F-labeled PET reagents and has mechanistic implications for ArBF(3)-enhanced ligandless metal-mediated cross coupling reactions with aryltrifluoroborates.

A DNAzyme with Three Protein-Like Functional Groups: Enhancing Catalytic Efficiency of M2+-Independent RNA Cleavage

Catalytically efficient, sequence-specific RNA cleavage holds great therapeutic value for selective gene inactivation against viral infection and cancer. Towards that end, SELEX and related combinatorial methods of in vitro selection have been used to discover RNA-cleaving DNAzymes that have received considerable attention. In addition to therapeutic use in catalysing sequence-specific destruction of mRNA for targeted gene deactivation, applications of catalysis to sensing have also been suggested. 9–26] Like ribozymes, DNAzymes present a limited chemical repertoire compared to proteins. 28] Nevertheless, this inherent lack of functionality can be offset by divalent metal cations (M ). For RNA cleavage, Mg + , or other divalent cations (M), afford kcat/KM values that approach the limits of catalytic perfection (10 m 1 min ) ; however, such values are observed only at high concentrations of Mg + (e.g. , 10–25 mm). In contrast, at physiological Mg concentrations (0.2–0.8 mm), even the most efficient of DNAzymes such as Dz10-23 and Dz8-17 exhibit markedly reduced kcat/KM values that fall in the range of 10–10 m 1 min . [33] These findings highlight an inextricable link between high concentrations of Mg and catalytic efficiency. 35] By the same token, one must hypothesise that the scarcity of intracellular Mg limits the efficacy of DNAzymes in vivo. This catalytic shortcoming might be circumvented by selecting for DNAzymes with synthetically appended chemical functionalities that enhance the chemical repertoire of nucleic acid enzymes, which is otherwise impaired by a low physiological concentration of M . The generation of functionalized nucleic acids for in vitro selection involves the enzymatic polymerization of synthetically functionalized nucleoside triphosphates (dXTPs in which X is any given nucleobase). An abiding interest in this approach is highlighted in numerous studies in which synthetic nucleotides have been successfully incorporated for the discovery of highly functionalized nucleic acids, aptamers, RNA-based enzymes 53] and DNAzymes. Our initial attempt at enhancing the chemical repertoire of DNAzymes resulted in the DNAzyme Dz925-11, which required both the imidazole and amine-bearing modified nucleotides 1 and 2 (Scheme 1) for activity. Nevertheless, catalytic properties remained modest: whereas a reasonably fast rate of selfcleavage was observed (kobs ~0.3 min ), the optimum temperature for self cleavage remained depressed at 13 8C; further, although the cis-cleaving DNAzyme was engineered to cleave at a single ribonucleoside linkage with both multiple turnover and high sequence specificity, 59] both optimal temperature (25 8C) and kcat (0.03 min ) also remained rather low. Notably, attempts to reselect 2nd-generation catalysts from either 20 or 40 degenerate positions failed to provide any improvement and instead Dz925-11 and closely related sequence variants embedded within the N40 region were selected. This “null result” led to an important conclusion: modified dNTPs 1 and 2 found in Dz925-11 failed to provide more efficient catalysts, even when selections were attempted from longer libraries composed of 40 nucleotides (N40). Consequently, we hypothesised that further increasing chemical diversity by introducing a third modified nucleotide bearing a cationic guanidinium ion would increase the number of viable catalytic solutions to M -independent RNA cleavage and afford DNAzymes that could operate at higher temperature with improved catalytic properties. Thus, the simultaneous polymerization of dATP (8-histaminyl-dATP), dUTP (5-guanidinoallyl-dUTP) and dCTP (5-aminoallyl-dCTP; 1, 3 and 4 in Scheme 1) and subsequent in vitro selection from a library of 20 degenerate positions led to the discovery of Dz9-86, which catalysed M-independent cleavage at a single ribo ACHTUNGTRENNUNG(cytosine)phosphodiester linkage at much higher temperatures (37–45 8C) with a rate constant similar to that of Dz925-11 (kobs~0.15 min ) under physiological conditions (200 mm MCl 0.2 mm Mg , 37 8C). Despite these quantitative and qualitative improvements, we expected room for improvement in terms of rate constants for self-cleavage and multiple turnover. In particular, exploration of greater Scheme 1. Chemical structures of 1 (dATP), 2 (dUTP), 3 (dUTP) and 4 (dCTP).

Novel Matrix Metalloproteinase Inhibitor [18F]Marimastat-Aryltrifluoroborate as a Probe for In vivo Positron Emission Tomography Imaging in Cancer

Matrix metalloproteinases (MMP), strongly associated pathogenic markers of cancer, have undergone extensive drug development programs. Marimastat, a noncovalent MMP inhibitor, was conjugated with FITC to label cellular metalloproteinase cancer targets in MDA-MB-231 cells in vitro. Punctate localization of active transmembrane MMP14 was observed. For molecular-targeted positron emission tomography imaging of syngeneic 67NR murine mammary carcinoma in vivo, marimastat was (18)F-labeled using a shelf-stable arylboronic ester conjugate as a captor for aqueous [(18)F]fluoride in a novel, rapid one-step reaction at ambient temperature. [(18)F]Marimastat-aryltrifluoroborate localized to the tumors, with labeling being blocked in control animals first loaded with >10-fold excess unlabeled marimastat. The labeled drug cleared primarily via the hepatobiliary and gastrointestinal tract, with multiple animals imaged in independent experiments, confirming the ease of this new labeling strategy.

Expanding the Catalytic Repertoire of Nucleic Acid Catalysts: Simultaneous Incorporation of Two Modified Deoxyribonucleoside Triphosphates Bearing Ammonium and Imidazolyl Functionalities

Two nucleoside triphosphates, a pyrimidine modified with an ammonium functionality and a purine modified with an imidazolyl functionality are compatible with all conditions for a combinatorial selection of nucleic-acid catalysts. We believe that this work is the first to demonstrate the potential for using not one but two modified nucleotides in tandem. The potential for an enriched catalytic repertoire is envisioned.

An Organotrifluoroborate for Broadly Applicable One‐Step 18F‐Labeling

A new zwitterionic organotrifluoroborate is appended to three radiosynthons that afford undergo facile bioconjugation to several clinically relevant peptides and one enzyme inhibitor. Molecularly complex bioconjugates are (18)F-labeled in a single aqueous step in rapid time (<15 min) without HPLC purification to afford tracers in good yields (>200 mCi, 20-40%) at high specific activity (≥3 Ci/μmol) and at >98% purity. PET imaging shows in vivo stability and tumor uptake.

Stoichiometric leverage: rapid 18F-aryltrifluoroborate radiosynthesis at high specific activity for click conjugation.

,values which are higher than those of most other radiotracers.Herein,weshowthefeasibilityofthismethodintermsofone-step labeling and click conjugation, along with experimentalproof of such high SAs.Propargylated arylborimidine 1 was synthesized accord-ing to previous reports as a shelf-stable B(dan)-protected

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 M(2+)-independent cleavage of an all-RNA target sequence (kobs = 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 (kobs = 0.27 min(-1)) than Dz9-86 (kobs = 0.13 min(-1)). The sequence composition of Dz12-91 bears a marked resemblance to that of Dz9-86 (kobs = 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 M(2+)-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.

Preclinical Evaluation of a High-Affinity 18F-Trifluoroborate Octreotate Derivative for Somatostatin Receptor Imaging

Recent studies have highlighted the high sensitivity of PET imaging with 68Ga-labeled octreotide derivatives for the detection and staging of neuroendocrine tumors. A somatostatin receptor ligand that is easily radiolabeled with 18F-fluoride could improve the availability of PET imaging of neuroendocrine tumors. We report an alkyltrifluoroborate–octreotate conjugate that is radiolabeled in a 1-step 18F exchange reaction in high yield and with high specific activity. Methods: We conjugated a new alkyltrifluoroborate to octreotate to obtain AMBF3-TATE, which was stored in 50-nmol aliquots for radiolabeling. 18F labeling was performed by 18F-19F isotope exchange with 18F-fluoride, and the tracer was purified by C18 cartridge separation. The radiochemical yield was 20%–25%. PET imaging and biodistribution were performed on mice bearing AR42J tumor xenografts. Results: AMBF3-TATE bound the somatostatin receptor subtype 2 with high affinity (inhibition constant, 0.13 ± 0.03 nM). Starting with 29.6–37 GBq (0.8–1 Ci) of 18F-fluoride, more than 7.4 GBq (>200 mCi) of 18F-AMBF3-TATE were obtained in 25 min (n = 5) with greater than 99% radiochemical purity at high specific activity (>111 GBq [3 Ci]/μmol). 18F-AMBF3-TATE was stable in plasma. PET imaging and biodistribution showed rapid renal excretion with low liver activity. High tumor uptake (10.11% ± 1.67% injected dose/g, n = 5) was detected at 60 min after injection. Bone uptake was negligible. Tumor-to-liver, tumor-to-blood, tumor-to-muscle, and tumor-to-bone ratios (at 60 min) were 26.2 ± 0.8, 25.1 ± 1.0, 89.0 ± 3.1, and 21.3 ± 3.6, respectively. Conclusion: 18F-AMBF3-TATE was radiolabeled in high yield and at high specific activity, did not require high-performance liquid chromatography purification, exhibited unexpectedly high binding affinity to somatostatin receptor subtype 2, and showed excellent pharmacokinetic properties in vivo, with high tumor uptake and high contrast ratios.