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Guanidinoglycosides are a class of non-cytotoxic molecular transporters capable of delivering high molecular weight bioactive cargos into cells at low nanomolar concentrations. Efficient bioconjugation with guanidinoglycosides has been previously demonstrated by utilizing a guanidinoneomycin decorated with a reactive but also unstable N-hydroxysuccinimmide ester-containing linker. Herein we report the synthesis, chemistry, and application of a new, stable guanidinoneomycin derivative armed with a highly specific maleimide moiety which allows for thiol-maleimide click chemistry, a highly popular bioconjugation strategy, widening the field of application of these intriguing and useful delivery vehicles.

The first generation of CB(1) positive allosteric modulators (e.g., ZCZ011) featured a 3-nitroalkyl-2-phenyl-indole structure. Although a small number of drugs include the nitro group, it is generally not regarded as being “drug-like”, and this is particularly true for aliphatic nitro groups. There are very few case studies where an appropriate bioisostere replaced a nitro group that had a direct role in binding. This may be indicative of the difficulty of replicating its binding interactions. Herein, we report the design and synthesis of ligands targeting the allosteric binding site on the CB(1) cannabinoid receptor, in which a CF(3) group successfully replaced the aliphatic NO(2). In general, the CF(3)-bearing compounds were more potent than their NO(2) equivalents and also showed improved in vitro metabolic stability. The CF(3) analogue (1) with the best balance of properties was selected for further pharmacological evaluation. Pilot in vivo studies showed that (±)-1 has similar activity to (±)-ZCZ011, with both showing promising efficacy in a mouse model of neuropathic pain.

The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels-Alder (IEDDA) reaction is the fastest bioorthogonal "click"ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a-11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5-11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.

Fatty acid oxidation (FAO) produces most of the ATP used to sustain the cardiac contractile work, although glycolysis is a secondary source of ATP under normal physiological conditions. FAO impairment has been reported in the advanced stages of heart failure (HF) and is strongly linked to disease progression and severity. Thus, from a clinical perspective, FAO dysregulation provides prognostic value for HF progression, the assessment of which could be used to improve patient monitoring and the effectiveness of therapy. Positron emission tomography (PET) imaging represents a powerful tool for the assessment and quantification of metabolic pathwaysin vivo. Several FAO PET tracers have been reported in the literature, but none of them is in routine clinical use yet. Metabolically trapped tracers are particularly interesting because they undergo FAO to generate a radioactive metabolite that is subsequently trapped in the mitochondria, thus providing a quantitative means of measuring FAOin vivo. Herein, we describe the design, synthesis, tritium labelling and radiofluorination of 4,4,16-trifluoro-palmitate (1) as a novel potential metabolically trapped FAO tracer. Preliminary PET-CT studies on [F-18]1in rats showed rapid blood clearance, good metabolic stability - confirmed by using [H-3]1in vitro - and resistance towards defluorination. However, cardiac uptake in rats was modest (0.24 +/- 0.04 % ID/g), and kinetic analysis showed reversible uptake, thus indicating that [F-18]1is not irreversibly trapped.

PFKFB3, a glycolysis-related enzyme upregulated in inflammatory conditions and angiogenesis, is an emerging target for diagnosis and therapy of atherosclerosis. The fluorinated phenoxindazole [18F]ZCDD083was synthesized, radiolabeled in 17 ± 5% radiochemical yield and >99% radiochemical purity, and formulated for preclinical PET/CT imaging in mice. In vivo stability analysis showed no significant metabolite formation. Biodistribution studies showed high blood pool activity and slow hepatobiliary clearance. Significant activity was detected in the lung 2 h postinjection (pi) (11.0 ± 1.5%ID/g), while at 6 h pi no pulmonary background was observed. Ex vivo autoradiography at 6 h pi showed significant high uptake of [18F]ZCDD083 in the arch region and brachiocephalic artery of atherosclerotic mice, and no uptake in control mice, matching plaques distribution seen by lipid staining along with PFKFB3 expression seen by immunofluorescent staining. In vivo PET scans showed higher aortic region uptake of [18F]ZCDD083in atherosclerotic ApoE–/–Fbn1C1039G+/− than in control mice (0.78 ± 0.05 vs 0.44 ± 0.09%ID/g). [18F]ZCDD083 was detected in aortic arch and brachiocephalic artery of ApoE–/– (with moderate atherosclerosis) and ApoE–/–Fbn1C1039G+/− (with severe, advanced atherosclerosis) mice, suggesting this tracer may be useful for the noninvasive detection of atherosclerotic plaques in vivo.

Prostate cancer represents a major public health threat as it is one of the most common male cancers worldwide. The prostate-specific membrane antigen (PSMA) is highly over-expressed in prostatic cancer cells in a manner that correlates with both tumour stage and clinical outcome. As such, PSMA has been identified as an attractive target for both imaging and treatment of prostate cancer. In recent years the focus on urea-based peptidomimetic inhibitors of the PSMA (representing low molecular weight/high affinity binders) has intensified as they have found use in the clinical imaging of prostate tumours. Reported herein are the design, synthesis and evaluation of a new fluorinated PSMA targeting small-molecule, FDA-PEG-GUL, which possesses the Glu-NH-CO-NH-Lys pharmacophore conjugated to a 5'-fluorodeoxy- adenosine unit. Inhibition assays were performed with FDA-PEG-GUL which revealed that it inhibits the PSMA in the nanomolar range. Additionally, it has been purposely designed so that it can be produced using the fluorinase enzyme from its chlorinated precursor, allowing for the enzymatic synthesis of radiolabelled [F-18]FDA-PEG-GUL via a nucleophilic reaction that takes place in experimentally advantageous conditions (in water at neutral pH and at ambient temperature). Specific binding of [F-18]FDA-PEG-GUL to PSMA expressing cancer cells was demonstrated, validating it as a promising PSMA diagnostic tool. This work establishes a successful substrate scope expansion for the fluorinase and demonstrates its first application towards targeting the PSMA.

We thank the Engineering and Physical Sciences Research Council, UK, for a research grant. We report a last-step fluorinase-catalyzed [18F]-fluorination of a cysteine-containing RGD peptide. The peptide was attached through sulfur to a modified and more hydrophilic variant of the recently disclosed Barbas linker which was itself linked to a chloroadenosine moiety via a PEGylated chain. The fluorinase was able to use this construct as a substrate for a transhalogenation reaction to generate [18F]-radiolabeled RGD peptides, which retained high affinity to cancer-cell relevant αvβ3 integrins. Postprint

The discovery of liquid water at several locations in the solar system raises the possibility that microbial life may have evolved outside Earth and as such could be accidently introduced into the Earth&rsquo, s ecosystem. Unusual sugars or amino acids, like non-proteinogenic isovaline and &alpha, aminoisobutyric acid that are vanishingly rare or absent from life forms on Earth, have been found in high abundance on non-terrestrial carbonaceous meteorites. It is therefore conceivable that exo-microorganisms might contain proteins that include these rare amino acids. We therefore asked whether the mammalian immune system would be able to recognize and induce appropriate immune responses to putative proteinaceous antigens that include these rare amino acids. To address this, we synthesised peptide antigens based on a backbone of ovalbumin and introduced isovaline and &alpha, aminoisobutyric acid residues and demonstrated that these peptides can promote na&iuml, ve OT-I cell activation and proliferation, but did so less efficiently than the canonical peptides. This is relevant to the biosecurity of missions that may retrieve samples from exoplanets and moons that have conditions that may be permissive for life, suggesting that accidental contamination and exposure to exo-microorganisms with such distinct proteomes might pose an immunological challenge.

The use of radiolabelled antibodies and antibody-derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin-avidin/streptavidin pairing, along with the inverse-electron-demand Diels-Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase-mediated transhalogenation [5'-chloro-5'-deoxyadenosine (ClDA) substrates to 5'-fluoro-5'-deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA-PEG-tetrazine and a [18 F]FDA-PEG-biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans-cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [18 F]FDA-PEG-biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase-catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C-2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [18 F]fluoride takes place in water at neutral pH and at ambient temperature.

Synthetic tubulysins 24a-m, containing non-hydrolysable N-substituents on tubuvaline (Tuv), were obtained in high purity and good overall yields using a multistep synthesis. A key step was the formation of differently N-substituted Ile-Tuv fragments 10 by using an aza-Michael reaction of azido-Ile derivatives 8 with the ,-unsaturated oxo-thiazole 5. A structure-activity relationship study using a panel of human tumour cell lines showed strong anti-proliferative activity for all compounds 24a-m, with IC50 values in the sub-nanomolar range, which were distinctly lower than those of tubulysinA, vinorelbine and paclitaxel. Furthermore, 24a-m were able to overcome cross-resistance to paclitaxel and vinorelbine in two tumour cell lines with acquired resistance to doxorubicin. Compounds 24e and 24g were selected as leads to evaluate their mechanism of action. In vitro assays showed that both 24e and 24g interfere with tubulin polymerization in a vinca alkaloid-like manner and prevent paclitaxel-induced assembly of tubulin polymers. Both compounds exerted antimitotic activity and induced apoptosis in cancer cells at very low concentrations. Compound 24e also exhibited potent antitumor activity at well tolerated doses on in vivo models of diffuse malignant peritoneal mesothelioma, such as MESOII peritoneal mesothelioma xenografts, the growth of which was not significantly affected by vinorelbine. These results indicate that synthetic tubulysins 24 could be used as standalone chemotherapeutic agents in difficult-to-treat cancers.

Non-peptidic RGD-mimic ligands were designed and synthesized by click chemistry between an arginine-azide mimic and an aspartic acid-alkyne mimic. Some of these molecules combine excellent in vitro properties (high αvβ3 affinity, selectivity, drug-like logD, high metabolic stability) with a variety of radiolabeling options (e.g. tritium and [18F]fluorine, plus compatibility with radio-iodination), not requiring the use of chelators or prosthetic groups. The binding mode of the resulting triazole RGD-mimics to αvβ3 or αIIbβ3 receptors was investigated by molecular modeling simulations. Compound 12 was successfully radiofluorinated and used for in vivo PET/CT studies in U87-tumour models, which showed only modest tumour uptake and retention, owing to rapid excretion. These results demonstrate that the novel click-RGD mimics are excellent radiolabeled probes for in vitro and cell-based studies on αvβ3 integrin, whereas further optimization of their pharmaco-kinetic and dynamic profile would be necessary for a successful use in in vivo imaging.

A novel class of cannabinoid ligands was synthesized in good overall yields by means of oxime-bio-conjugation between hydroxylamine-functionalized Rimonabant-type pyrazoles and fluoro-deoxy-carbohydrates (D-2-fluoro-deoxy-glucose, FDG, and D-5-fluoro-5-deoxy-ribose, FDR). FDR proved to be superior to FDG for bio-conjugation, as it occurred in milder conditions and at faster rate (rt, 20 min vs. 100 degrees C, 30 min). All of the title compounds showed relatively modest affinity for the CBI receptor (high nanomolar range) and selectivities vs. the CB2. (C) 2013 Elsevier B.V. All rights reserved.

Introduction 5-[ 18 F]Fluoro-5-deoxyribose ([ 18 F]FDR) 3 was prepared as a novel monosaccharide radiotracer in a two-step synthesis using the fluorinase, a C-F bond forming enzyme, and a nucleoside hydrolase. The resulting [ 18 F]FDR 3 was then explored as a radiotracer for imaging tumours (A431 human epithelial carcinoma) by positron emission tomography in a mice model. Methods 5-[ 18 F]Fluoro-5-deoxyribose ([ 18 F]FDR) 3 , was prepared by incubating S -adenosyl-L-methionine (SAM) and [ 18 F]fluoride with the fluorinase enzyme, and then incubating the product of this reaction, [ 18 F]-5'-fluoro-5'-deoxadenosine ([ 18 F]FDA) 2 , with an adenosine hydrolase to generate [ 18 F]FDR 3 . The enzymes were freeze-dried and were used on demand by dissolution in buffer solution. The resulting [ 18 F]FDR 3 was then administered to four mice that had tumours induced from the A431 human epithelial carcinoma cell line. Results The tumour (A431 human epithelial carcinoma) bearing mice were successfully imaged with [ 18 F]FDR 3 . The radiotracer displayed good tumour imaging resolution. A direct comparison of the uptake and efflux of [ 18 F]FDR 3 with 2-[ 18 F]fluoro-2-deoxyglucose ([ 18 F]FDG) was made, revealing comparative tumour uptake and imaging potential over the first 10–20min. The study revealed however that [ 18 F]FDR 3 does not accumulate in the tumour as efficiently as [ 18 F]FDG over longer time periods. Conclusions [ 18 F]FDR 3 can be rapidly synthesised in a two enzyme protocol and used to image tumours in small animal models.

Fenretinide (4-HPR) is a synthetic derivative of all-trans-retinoic acid (ATRA) characterised by improved therapeutic properties and toxicological profile relative to ATRA. 4-HPR has been mostly investigated as an anti-cancer agent, but recent studies showed its promising therapeutic potential for preventing metabolic syndrome. Several biological targets are involved in 4-HPR's activity, leading to the potential use of this molecule for treating different pathologies. However, although 4-HPR displays quite well-understood multitarget promiscuity with regards to pharmacology, interpreting its precise physiological role remains challenging. In addition, despite promising results in vitro, the clinical efficacy of 4-HPR as a chemotherapeutic agent has not been satisfactory so far. Herein, we describe the preparation of a library of 4-HPR analogues, followed by the biological evaluation of their anti-cancer and anti-obesity/diabetic properties. The click-type analogue 3 b showed good capacity to reduce the amount of lipid accumulation in 3T3-L1 adipocytes during differentiation. Furthermore, it showed an IC50 of 0.53±0.8 ?M in cell viability tests on breast cancer cell line MCF-7, together with a good selectivity (SI=121) over noncancerous HEK293 cells. Thus, 3 b was selected as a potential PET tracer to study retinoids in vivo, and the radiosynthesis of [18F]3b was successfully developed. Unfortunately, the stability of [18F]3b turned out to be insufficient to pursue imaging studies.