Design of Bifunctional Pyclen-Based Lanthanide Luminescent Bioprobes for Targeted Two-Photon Imaging.

In the past, Lanthanide Luminescent Bioprobes (LLBs) based on pyclen-bearing π-extended picolinate antennas were synthesized and demonstrated well-adapted optical properties for biphotonic microscopy. The objective of this work is to develop a strategy to design bifunctional analogues of the previously studied LLBs presenting an additional reactive chemical group to allow their coupling to biological vectors to reach deep in vivo targeted two-photon bioimaging. Herein, we elaborated a synthetic scheme allowing the introduction of a primary amine on the para position of the macrocyclic pyridine unit. The photophysical and bioimaging studies demonstrate that the introduction of the reactive function does not alter the luminescent properties of the LLBs paving the way for further applications.

Synthesis and Photophysical Properties of Lanthanide Pyridinylphosphonic Tacn and Pyclen Derivatives: From Mononuclear Complexes to Supramolecular Heteronuclear Assemblies.

Synthetic methodologies were developed to achieve the preparation of ligands L1 and L2 consisting of tacn- and pyclen-based chelators decorated with pyridinylphosphonic pendant arms combined with ethylpicolinamide or acetate coordinating functions, respectively. Phosphonate functions have been selected for their high affinity toward Ln3+ ions compared to their carboxylated counterparts and for their steric hindrance that favors the formation of less-hydrated complexes. Thanks to regiospecific N-functionalization of the macrocyclic backbones, the two ligands were isolated with good yields and implicated in a comprehensive photophysical study for the complexation of Eu3+, Tb3+, and Yb3+. The coordination behavior of L1 and L2 with these cations has been first investigated by means of a combination of UV-vis absorption spectroscopy, steady-state and time-resolved luminescence spectroscopy, and 1H and 31P NMR titration experiments. Structural characterization in solution was assessed by NMR spectroscopy, corroborated by theoretical calculations. Spectroscopic characterization of the Ln3+ complexes of L1 and L2 was done in water and D2O and showed the effective sensitization of the lanthanide metal-centered emission spectra, each exhibiting typical lanthanide emission bands. The results obtained for the phosphonated ligands were compared with those reported previously for the corresponding carboxylated analogues.

New Triazacycloalkane Derivatives as Cytotoxic Agents for CLL Treatment: From Proof of Concept to the Targeting Biomolecule.

The 1,4,7-tris-(2-pyridinylmethyl)-1,4,7-triazacyclononane ligand (no3py) and its bifunctional analogue no3pyCOOK were synthesized to investigate their action toward zinc(II) depletion related to the apoptosis phenomenon in chronic lymphocytic leukemia (CLL) cells. no3py was used as the "free" ligand, while its "graftable" derivative was conjugated on a newly synthesized bifunctional sialoglycan, 6'-SL-NH2, selected to specifically bind CD22 biomarker expressed on the B-CLL cell surface. Both compounds were produced with good yields thanks to a Sonogashira coupling reaction and an orthoester function, respectively, for the chelator and the targeting moiety. The newly reported bioconjugate 6'-SL-no3py was then obtained through a peptidic coupling reaction. Biological in vitro studies of no3py and 6'-SL-no3py consisting of real-time detection of cell health (cytotoxicity and proliferation) and caspases 3/7 activation (crucial enzymes whose activation triggers cell death signaling pathways) have been investigated. First, Ramos, Daudi, and Raji B-cell lines, which present different sensitivity to zinc(II) content variation, were incubated with no3py and 6'-SL-no3py. Then, a videomicroscope allowed the real-time monitoring of the morphological changes leading to cell death from the detection of the cytotoxicity, the antiproliferative effect, and the caspasic activity. In terms of mechanism, the Zn2+ chelator cytotoxic effect of no3py has been evidenced by a culture medium ion supplementation study and by the decrease of intracellular fluorescence of Zn-specific fluorophore zinquin in the presence of no3py and 6'-SL-no3py chelators. Finally, flow cytometry analysis with classical Annexin V staining was conducted to detect no3py- and 6'-SL-no3py-induced apoptotic cell death in B-CLL cells. Time-course analysis, using the Incucyte Live-Cell Analysis System, demonstrated that no3py induced cell death in a time- and dose-dependent manner with variability across cell lines. 6'-SL-no3py exhibited the same dose-dependent trend as no3py, showing the efficiency of the targeting moiety. In both cases, the chelators depicted proliferation curves that were inversely correlated with kinetic death. Morphological changes specific to apoptosis and caspase 3/7 activation were observed for the three cell lines treated with no3py and 6'-SL-no3py, highlighting their role as apoptotic agents. A higher concentration of 6'-SL-no3py is needed to reach 50% of the B-CLL mortality, confirming a targeting of the chelator to the cell membrane. Overall, our results proved that the biological properties of the triazamacrocyclic chelator still remain even after addition of the targeting moiety. The free chelator as well as the bioconjugate constitute promising cytotoxic agents for CLL therapy through apoptosis induction.

Pyclen-Based Ligands Bearing Pendant Picolinate Arms for Gadolinium Complexation.

We report the synthesis of two pyclen-based regioisomer ligands (pyclen = 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene) functionalized with picolinic acid pendant arms either at positions 3,9-pc2pa (L5) or 3,6-pc2pa (L6) of the macrocyclic fragment. The ligands were prepared by the regiospecific protection of one of the amine nitrogen atoms of the macrocycle using Boc and Alloc protecting groups, respectively. The X-ray structure of the Gd(III) complex of L5 contains trinuclear [(GdL5)3(H2O)3]3+ entities in which the monomeric units are joined by µ2-η1:η1-carboxylate groups. However, the 1H and 89Y NMR spectra of its Y(III) analogue support the formation of monomeric complexes in solution. The Tb(III) complexes are highly luminescent, with emission quantum yields of up to 28% for [TbL5]+. The luminescence lifetimes recorded in H2O and D2O solutions indicate the presence of a water molecule coordinated to the metal ion, as also evidenced by the 1H relaxivities measured for the Gd(III) analogues. The Gd(III) complexes present very different exchange rates of the coordinated water molecule (kex298 = 87.1 × 106 and 1.06 × 106 s-1 for [GdL5]+ and [GdL6]+, respectively). The very high water exchange rate of [GdL5]+ is associated with the steric hindrance originating from the coordination of the ligand around the water binding site, which favors a dissociatively activated water exchange process. The Gd(III) complexes present rather high thermodynamic stabilities (log KGdL = 20.47 and 19.77 for [GdL5]+ and [GdL6]+, respectively). Furthermore, these complexes are remarkably inert with respect to their acid-assisted dissociation, in particular the complex of L5.

Complexation of Mn(II) by Rigid Pyclen Diacetates: Equilibrium, Kinetic, Relaxometric, Density Functional Theory, and Superoxide Dismutase Activity Studies.

We report the Mn(II) complexes with two pyclen-based ligands (pyclen = 3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene) functionalized with acetate pendant arms at either positions 3,6 (3,6-PC2A) or 3,9 (3,9-PC2A) of the macrocyclic fragment. The 3,6-PC2A ligand was synthesized in five steps from pyclen oxalate by protecting one of the secondary amine groups of pyclen using Alloc protecting chemistry. The complex with 3,9-PC2A is characterized by a higher thermodynamic stability [log KMnL = 17.09(2)] than the 3,6-PC2A analogue [log KMnL = 15.53(1); 0.15 M NaCl]. Both complexes contain a water molecule coordinated to the metal ion, which results in relatively high 1H relaxivities (r1p = 2.72 and 2.91 mM-1 s-1 for the complexes with 3,6-PC2A and 3,9-PC2A, respectively, at 25 °C and 0.49 T). The coordinated water molecule displays fast exchange kinetics with the bulk in both cases; the rates (kex298) are 140 × 106 and 126 × 106 s-1 for [Mn(3,6-PC2A)(H2O)] and [Mn(3,9-PC2A)(H2O)], respectively. The two complexes were found to be remarkably inert with respect to their dissociation, with half-lives of 63 and 21 h, respectively, at pH = 7.4 in the presence of excess Cu(II). The r1p values recorded in blood serum remain constant at least over a period of 120 h. Cyclic voltammetry experiments show irreversible oxidation features shifted to higher potentials with respect to [Mn(EDTA)(H2O)]2- (H4EDTA = ethylenediaminetetraacetic acid) and [Mn(PhDTA)(H2O)]2- (H4PhDTA = phenylenediamine-N,N,N',N'-tetraacetic acid), indicating that the PC2A complexes reported here have a lower tendency to stabilize Mn(III). The superoxide dismutase activity of the Mn(II) complexes was tested using the xanthine/xanthine oxidase/p-nitro blue tetrazolium chloride assay at pH = 7.8. The Mn(II) complexes of 3,6-PC2A and 3,9-PC2A are capable of assisting decomposition of the superoxide anion radical. The kinetic rate constant of the complex of 3,9-PC2A is smaller by 1 order of magnitude than that of 3,6-PC2A.

Pyclen-Based Ln(III) Complexes as Highly Luminescent Bioprobes for In Vitro and In Vivo One- and Two-Photon Bioimaging Applications.

In addition to the already described ligand L4a, two pyclen-based lanthanide chelators, L4b and L4c, bearing two specific picolinate two-photon antennas (tailor-made for each targeted metal) and one acetate arm arranged in a dissymmetrical manner, have been synthesized, to form a complete family of lanthanide luminescent bioprobes: [EuL4a], [SmL4a], [YbL4b], [TbL4c], and [DyL4c]. Additionally, the symmetrically arranged regioisomer L4a' was also synthesized as well as its [EuL4a'] complex to highlight the astonishing positive impact of the dissymmetrical N-distribution of the functional chelating arms. The investigation clearly shows the high performance of each bioprobe, which, depending on the complexed lanthanide, could be used in various applications. Each presents high brightness, quantum yields, and lifetimes. Staining of the complexes into living human breast cancer cells was observed. In addition, in vivo two-photon microscopy was performed for the first time on a living zebrafish model with [EuL4a]. No apparent toxicity was detected on the growth of the zebrafish, and images of high quality were obtained.

Synthesis and biological evaluation of phosphoramidate prodrugs of two analogues of 2-deoxy-d-ribose-1-phosphate directed to the discovery of two carbasugars as new potential anti-HIV leads.

2-Deoxy-α-d-ribose-1-phosphate is of great interest as it is involved in the biosynthesis and/or catabolic degradation of several nucleoside analogues of biological and therapeutic relevance. However due to the lack of a stabilising group at its 2-position, it is difficult to synthesize stable prodrugs of this compound. In order to overcome this lack of stability, the synthesis of carbasugar analogues of 2-deoxyribose-1-phosphate was envisioned. Herein the preparation of a series of prodrugs of two carbocyclic analogues of 2-deoxyribose-1-phosphate using the phosphoramidate ProTide technology, along with their biological evaluation against HIV and cancer cell proliferation, is reported.

Synthesis, anti-HIV and cytostatic evaluation of 3'-deoxy-3'-fluorothymidine (FLT) pro-nucleotides.

A series of pro-nucleotide phosphoramidates and phosphorodiamidates of the antiviral lead compound 3'-deoxy-3'-fluorothymidine (FLT) have been designed and synthesized. In vitro antiretroviral and cytostatic studies revealed potent (sub-micromolar) inhibition of HIV-1 and HIV-2 replication, with retention of activity in thymidine kinase-negative cell models, as predicted by the ProTide concept.

Synthesis and biological evaluation of prodrugs of 2-fluoro-2-deoxyribose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate.

We report in this Letter the synthesis of prodrugs of 2-fluoro-2-deoxyarabinose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate. We demonstrate the difficulty of realising a phosphorylation step on the anomeric position of 2-deoxyribose, and we discover that introduction of fluorine atoms on the 2 position of 2-deoxyribose enables the phosphorylation step: in fact, the stability of the prodrugs increases with the degree of 2-fluorination. Stability studies of produgs of 2-fluoro-2-deoxyribose-1-phosphate and 2,2-difluoro-2-deoxyribose-1-phosphate in acidic and neutral conditions were conducted to confirm our observation. Biological evaluation of prodrugs of 2,2-difluoro-2-deoxyribose-1-phosphate for antiviral and cytotoxic activity is reported.

Synthesis of the natural enantiomer of neplanocin B.

(-)-Neplanocin B, the natural isomer of a component of the neplanocin family was diasteroselectively synthesized from 2,3-O-isopropylidene-D-1,4-ribonolactone. However, when evaluated against several DNA and RNA viruses in cell culture experiments, it did not show any antiviral activity.

Synthesis of 3'-halo-5'-norcarbocyclic nucleoside phosphonates as potent anti-HIV agents.

The synthesis and the antiviral evaluation of 3'-halo (iodo and fluoro) 5'-norcarbocyclic nucleoside phosphonates is described. No antiviral activity was observed against Zika virus, Dengue virus 2, HSV-1, HSV-2 and Chikungunya virus. In contrast, some of the synthesized compounds are potent inhibitors of the replication of HIV-1, comparatively to (R)-PMPA, with no concomitant cytotoxicity.

Combining a pyclen framework with conjugated antenna for the design of europium and samarium luminescent bioprobes.

The first pyclen based ligand bearing two picolinate intra-ligand charge transfer transition antennae and one acetate arm organized in a dissymmetric manner was synthesized for Eu(iii) and Sm(iii) complexation. The europium complex presents an excellent brightness and biphotonic imaging of T24-cells has been performed using Eu(iii) and the less common Sm(iii) bioprobes.

DNA photochemistry: geometrically unconstrained pyrimidine (6-4) pyrimidone photoproducts do photoisomerize.

Structural features are of major importance for the formation of mutagenic photoproducts in DNA. It was recently reported that lack of constraints between two adjacent nucleosidic units prevents the conversion of pyrimidine (6-4) pyrimidone photoproducts into their Dewar valence isomers. We here report that this is not the case for the thymidine photoproducts which, although unconstrained, are quantitatively converted into photolysis products identified as Dewar valence isomers by mass spectrometry and NMR and infrared spectroscopies.

A guanine-ethylthioethyl-glutathione adduct as a major DNA lesion in the skin and in organs of mice exposed to sulfur mustard.

Sulfur mustard (SM) is an old chemical warfare but it remains a threat to both militaries and civilians. SM mainly targets skin, eyes and lungs and diffuses to internal organs. At the molecular level, SM is able to damage DNA through the formation of monoadducts and biadduct. Glutathione (GSH) is another critical target of SM in cells since it is part of the detoxification mechanism against alkylating agents. In the present work, we investigated whether SM could form covalent bonds simultaneously with a DNA base and the sulfhydryl group of GSH. The expected guanine adduct, S-[2-(N7-guanyl)-ethylthioethyl]-glutathione (N7Gua-ETE-GSH), was synthesized and detected in several tissues of SKH-1 mice exposed to 60mg/kg of SM in the dorsal-lumbar region. N7Gua-ETE-GSH was detected in all organs studied, except in the liver. The tissue exhibiting the highest levels of N7Gua-ETE-GSH was skin, followed by brain, lungs, kidneys and spleen. N7Gua-ETE-GSH was detected in skin, brain and lungs as long as two weeks after exposure. The persistence was less in other organs. The observation of the formation of N7Gua-ETE-GSH in vivo confirms the variety of damages induced by SM in DNA. It also provides another example of the formation of DNA adducts involving glutathione following in vivo exposure to bifunctional alkylating compounds.

Design, synthesis and biological evaluation of phosphorodiamidate prodrugs of antiviral and anticancer nucleosides.

We herein report the application of the phosphorodiamidate phosphate prodrug approach to a series of thirteen nucleoside analogs with antiviral or anticancer activity. Twenty-five symmetrical phosphorodiamidates were synthesized, bearing esterified l-Alanine (and in one case d-Alanine) in the prodrug moiety, each as single stereoisomer. The presence of an achiral phosphorus represents a potential advantage over the phosphoramidate ProTide approach, where diastereoisomeric mixtures are routinely obtained, and different biological profiles may be expected from the diastereoisomers. Optimization of the synthetic pathway allowed us to identify two general methods depending on the particular nucleoside analogs. All the compounds were biologically evaluated in antiviral and anticancer assays and several showed improvement of activity compared to their parent nucleosides, as in the case of ddA, d4T, abacavir and acyclovir against HIV-1 and/or HIV-2. The biological results were supported by metabolism studies with carboxypeptidase Y monitored by (31)P NMR to investigate their bioactivation. This work further validates the phosphorodiamidate approach as a monophosphate prodrug motif with broad application in the antiviral and anticancer fields.

First enantioselective synthesis of (-)-neplanocin B.

(-)-Neplanocin B, the natural isomer of a component of the neplanocin family was enantioselectively synthesized.