Synthesis and activity of fluorescent isoprenoid pyrophosphate analogues

New fluorescent analogues of farnesol and geranylgeraniol have been prepared and then converted to the corresponding pyrophosphates. These analogues incorporate anthranylate or dansyl-like groups anchored to the terpenoid skeleton through amine bonds that would be expected to be relatively stable to metabolism. After addition of the alcohols or the pyrophosphates to the culture medium, their fluorescence is readily observed inside a human-derived leukemia cell line. Enzyme assays have revealed that the farnesyl pyrophosphate analogue is an inhibitor of FTase, while the corresponding alcohol is not. These results, together with Western blot analyses of cell lysates, indicate that the farnesyl pyrophosphate analogue penetrates the cells as an intact pyrophosphate and that it does so at a biologically relevant concentration.     

Synthesis and enzyme inhibitory activity of the s-nucleoside analogue of the ribitylaminopyrimidine substrate of lumazine synthase and product of riboflavin synthase

Lumazine synthase and riboflavin synthase catalyze the last two steps in the biosynthesis of riboflavin. To obtain structural and mechanistic probes of these two enzymes, as well as inhibitors of potential value as antibiotics, a sulfur analogue of the pyrimidine substrate of the lumazine synthase-catalyzed reaction and product of the riboflavin synthase-catalyzed reaction was designed. Facile syntheses of the S-nucleoside 5-amino-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione hydrochloride (15) and its nitro precursor 5-nitro-6-(D-ribitylthio)pyrimidine-2,4(1H,3H)-dione (14) are described. These compounds were tested against lumazine synthase and riboflavin synthase obtained from a variety of microorganisms. Compounds 14 and 15 were found to be inhibitors of both riboflavin synthase and lumazine synthase. Compound 14 is an inhibitor of Bacillus subtilis lumazine synthase (Ki 26 microM), Schizosaccharomyces pombe lumazine synthase (Ki 2.0 microM), Mycobacterium tuberculosis lumazine synthase (Ki 11 microM), Escherichia coli riboflavin synthase (Ki 2.7 microM), and Mycobacterium tuberculosis riboflavin synthase (Ki 0.56 muM), while compound 15 is an inhibitor of B. subtilis lumazine synthase (Ki 2.6 microM), S. pombe lumazine synthase (Ki 0.16 microM), M. tuberculosis lumazine synthase (Ki 31 microM), E. coli riboflavin synthase (Ki 47 microM), and M. tuberculosis riboflavin synthase (Ki 2.5 microM).     

Synthesis and characterization of a cell-permeable near-infrared fluorescent deoxyglucose analogue for cancer cell imaging

We report the synthesis and characterization of a novel NIR fluorescent deoxyglucose analogue, CyNE 2-DG. Experiments in different cell lines showed a preferential uptake of CyNE 2-DG in cancer cells and its effective competition with unlabeled d-glucose. Cell imaging experiments demonstrated the superior cell-permeability of CyNE 2-DG over the NIR standard IRDye 800CW 2-DG, and validated its application for cancer cell imaging in the NIR region.     

Synthesis of a conformationally restricted substrate analogue of siderophore biosynthetases

[reaction: see text] A conformationally restricted analogue (5) of N(omega)-acetyl-N(omega)-hydroxyornithine and -lysine was synthesized. The synthesis features an efficient acylnitroso hetero-Diels-Alder cycloadduct (1) ring opening with palladium(0) and methylnitroacetate.     

Synthesis of a fluorous ligand and its application for asymmetric addition of dimethylzinc to aldehydes

A new fluorous ligand was synthesized from the acetonide of dimethyl tartarate, which showed excellent asymmetric induction on the addition of dimethylzinc to aldehydes. This ligand will be useful for synthesis of bioactive compounds with a methyl carbinol moiety. It could be recycled without using a fluorous solvent or a fluorous column.     

Synthesis and evaluation of chlorinated substrate analogues for farnesyl diphosphate synthase

Substrate analogues for isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where the C3 methyl groups were replaced by chlorine, were synthesized and evaluated as substrates for avian farnesyl diphosphate synthase (FPPase). The IPP analogue (3-ClIPP) was a cosubstrate when incubated with dimethylallyl diphosphate (DMAPP) or geranyl diphosphate (GPP) to give the corresponding chlorinated analogues of geranyl diphosphate (3-ClGPP) and farnesyl diphosphate (3-ClFPP), respectively. No products were detected in incubations of 3-ClIPP with 3-ClDMAPP. Incubation of IPP with 3-ClDMAPP gave 11-ClFPP as the sole product. Values of K(M)(3-ClIPP) (with DMAPP) and K(M)(3-ClDMAPP) (with IPP) were similar to those for IPP and DMAPP; however, values of k(cat) for both analogues were substantially lower. These results are consistent with a dissociative electrophilic alkylation mechanism where the rate-limiting step changes from heterolytic cleavage of the carbon-oxygen bond in the allylic substrate to alkylation of the double bond of the homoallylic substrate.     

Design and synthesis of a transferable farnesyl pyrophosphate analogue to Ras by protein farnesyltransferase

The posttranslational addition of a farnesyl moiety to the Ras oncoprotein is essential for its membrane localization and is required for both its biological activity and ability to induce malignant transformation. We describe the design and synthesis of a farnesyl pyrophosphate (FPP) analogue, 8-anilinogeranyl pyrophosphate 3 (AGPP), in which the omega-terminal isoprene unit of the farnesyl group has been replaced with an aniline functionality. The key steps in the synthesis are the reductive amination of the alpha,beta-unsaturated aldehyde 5 to form the lipid analogue 6, and the subsequent conversion of the allylic alcohol 7 to the chloride 8 via Ph(3)PCl(2) followed by displacement with [(n-Bu)(4)N](3)HP(2)O(7) to give AGPP (3). AGPP is a substrate for protein farnesyltransferase (FTase) and is transferred to Ras by FTase with the same kinetics as the natural substrate, FPP. AGPP is highly selective, showing little inhibitory activity against either geranylgeranyl-protein transferase type I (GGTase I) (K(i) = 0.06 microM, IC(50) = 20 microM) or squalene synthase (IC(50) = 1000 microM). AGPP is the first efficiently transferable analogue of FPP to be modified at the omega-terminus that provides a platform from which additional analogues can be made to probe the biological function of protein farnesylation. AGPP is the first example of a class of compounds that are alternate substrates for protein isoprenylation that are not inhibitors of squalene synthase.     

Synthesis and resolution of a novel chiral diamine ligand and application to asymmetric lithiation-substitution

A short, efficient synthesis of chiral 1,5-diaza-cis-decalins (7) is presented. In the lithiation of N-Boc pyrrolidine, the ligands with the smallest most electron rich R groups (Me > Et > CH2tBu > CH2CF3 approximately Bn) were most effective. In the asymmetric deprotonation/substitution of benzylic substrates, (R,R)-7 (R = Me, R' = H) conferred modest selectivity. The ready availability of both enantiomers of the 1,5-diaza-cis-decalins and the ability to tune steric and electronic properties renders these compounds an attractive new class of diamine ligands.     

Human farnesyl pyrophosphate synthase inhibition by nitrogen bisphosphonates: a 3D-QSAR study

We report the results of a comparative molecular field analysis and comparative molecular similarity index analysis of the human farnesyl pyrophosphate synthase (FPPS) inhibition by nitrogen bisphosphonates (NBPs) taking into account their time-dependent inhibition efficacies. The 3D-QSAR models obtained provide steric, electrostatic and hydrophobic contour maps consistent with the interactions into the active site of human FPPS observed in available crystallographic structures. Furthermore, the 3D-QSAR models obtained provide accurately IC₅₀ values of the NBPs of the training set. The predictive ability of these 3D-QSAR models was found to rely on the choice of biologically active conformations of the target molecules and on a careful examination of the protonation status of the NBPs in the training set. The best models obtained can be useful to predict biological values of a high number of NBPs that have been used for the treatment of different diseases as potential inhibitors of the activity of the FPPS enzyme.     

Synthesis and infrared and fluorescent spectra of rare earth complexes with a new amide ligand

Solid complexes of rare earth nitrates and picrates with a new amide ligand, 1,6-bis[(2'-benzylaminoformyl)phenoxyl]hexane (L) have been prepared. These complexes are characterized by elemental analysis, UV-vis spectra and IR spectra. The fluorescent and luminescent properties of the Eu(III) and Tb(III) nitrates and picrates complexes in solid state are also investigated. Under the excitation of UV light, these complexes except Tb(III) picrate complex exhibit characteristic emission of europium and terbium ions. The influence of the counter anion on the fluorescent intensity is also discussed.     

Colorimetric and orange light-emitting fluorescent probe for pyrophosphate in water

A dual-mode probe based on a benzothiazolium hemicyanine chromophore was designed and synthesized for the detection of pyrophosphate (PPi) in water. The use of a fluorescent probe for colorimetric and long-wavelength fluorescence detection of PPi could be suitable for both rapid in-field and bioimaging experiments.     

Synthesis and photophysical evaluation of charge neutral thiourea or urea based fluorescent PET sensors for bis-carboxylates and pyrophosphate

The synthesis of the fluorescent photoinduced electron transfer (PET) chemosensors 1-3 for bis-anions such as bis-carboxylates and pyrophosphate in organic solvents is described herein. These sensors are based on the receptor-spacer-fluorophore-spacer-receptor motif where the receptors are charge neutral aromatic thiourea or urea receptors and the fluorophore is anthracene. The anion recognition was evaluated using 1H NMR as well as absorption and fluorescence spectroscopy in DMSO. For simple anions such as acetate or fluoride, the recognition was shown to be through hydrogen bonding of the corresponding anion to the receptors. This gave rise to only minor changes in the absorption spectra, but significant changes in the fluorescence emission spectra, which was substantially (70-95%) quenched. Analysis of these recognition events implied a 1 : 2 (sensor : anion) binding and ideal PET behaviour for ions such as AcO- and H2PO4-. For F-, the luminescent quenching indicated a 1 : 1 binding, but we deduced that this was due more to complete quenching of the excited state after the addition of one equivalent of the anion. For all of the anions, the quenching contributed to enhanced efficiency of PET from the receptors to the excited state of the fluorophore. In the case of the bis-anions (ambient), such as di-carboxylates, similar fluorescence quenching was observed. However, here either a 1 : 1 or a 1 : 2 binding was observed depending on the length of the spacer separating the two carboxylate moieties and the nature of the receptor. Whereas both pyrophosphate and malonate gave rise to a 1 : 1 binding, glutarate gave rise to approximately 1 : 2 binding for the thiourea sensors 1 and 2. However, for the urea based sensor 3, the binding was found to be 1 : 1 for all the bis-anions. For such a 1 : 1 binding we propose that the anion most likely bridges the fluorophore moiety. This was also evident from the 1H NMR (DMSO-d6) spectrum where the anthracene resonances were significantly affected. By simply modifying the electronic structure of the receptor, the sensitivity of the recognition process could also be modified; e.g. compound 1, bearing the trifluoromethyl substituent, showed stronger binding to the bis-anions than 2, which possessed a simple phenyl moiety.     

Dimerization and anion binding of a fluorescent phospholipid analogue

A diarylacetylene fluorophore featuring spatially separated urea and phosphocholine (PC) groups forms a macrocyclic "head-to-tail" dimer stabilized by NH(urea)···OP(PC) hydrogen bonds. At concentrations above ～2 × 10(-5) M in CH(2)Cl(2), the emission intensity of the dimer is quenched by HCO(3)(-) and H(2)PO(4)(-) but not by Cl(-) and NO(3)(-). Under more dilute conditions, all four anions are bound unselectively with association constants on the order of 10(5) M(-1).     

Application of a green fluorescent fusion protein to study protein-protein interactions by electrophoretic methods

A screening procedure for protein-protein interactions in cellular extracts using a green fluorescent protein (GFP) and affinity capillary electrophoresis (ACE) was established. GFP was fused as a fluorescent indicator to the C-terminus of a cyclophilin (rDmCyp20) from Drosophila melanogaster. Cyclophilins (Cyps) belong to the ubiquitously distributed enzyme family of peptidyl-prolyl cis/trans isomerases (PPlases) and are well known as cellular targets of the immunosuppressive drug cyclosporin A (CsA). The PPlase activity of the GFP fused rDmCyp20 as well as the high affinity to CsA remain intact. Using native gel electrophoresis and ACE mobility-shift assays, it was demonstrated that the known moderate affinity of Cyp20 to the capsid protein p24 of HIV-1 was detectable in the case of rDmCyp20 fused to the fluorescent tag. For the p24 / rDmCyp20-GFP binding an ACE method was established which allowed to determine a dissociation constant of Kd = 20+/-1.5 x 10(-6) M. This result was verified by size-exclusion chromatography and is in good agreement with published data for the nonfused protein. Moreover the fusion protein was utilized to screen rDmCyp20-protein interactions by capillary electrophoresis in biological matrices. A putative ligand of rDmCyp20 in crude extracts of embryonic D. melanogaster was discovered by mobility-shift assays using native gel electrophoresis with fluorescence imaging and ACE with laser-induced fluorescence detection. The approach seems applicable to a wide range of proteins and offers new opportunities to screen for moderate protein-protein interactions in biological samples.     

Crystallographic studies on the reaction of isopenicillin N synthase with an unsaturated substrate analogue

Isopenicillin N synthase (IPNS) catalyses conversion of the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N (IPN), the central step in biosynthesis of the beta-lactam antibiotics. The unsaturated substrate analogue delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-vinylglycine (ACvG) has previously been incubated with IPNS and single product was isolated, a 2-alpha-hydroxymethyl isopenicillin N (HMPen), formed via a monooxygenase mode of reactivity. ACvG has now been crystallised with IPNS and the structure of the anaerobic IPNS:Fe(II):ACvG complex determined to 1.15 A resolution. Furthermore, by exposing the anaerobically grown crystals to high-pressure oxygen gas, a structure corresponding to the bicyclic product HMPen has been obtained at 1.60 A resolution. In light of these and other IPNS structures, and recent developments with related dioxygenases, the [2 + 2] cycloaddition mechanism for HMPen formation from ACvG has been revised, and a stepwise radical mechanism is proposed. This revised mechanism remains consistent with the observed stereospecificity of the transformation, but fits better with apparent constraints on the coordination geometry around the active site iron atom.     

Synthesis and preliminary cytotoxicity study of a cephalosporin-CC-1065 analogue prodrug

Background  

Antibody-directed enzyme prodrug therapy (ADEPT) is a promising new approach to deliver anticancer drugs selectively to tumor cells. In this approach, an enzyme is conjugated to a tumor-specific antibody. The antibody selectively localizes the enzyme to the tumor cell surface. Subsequent administration of a prodrug substrate of the enzyme leads to the enzyme-catalyzed release of the free drug at the tumor site. The free drug will destroy the tumor cells selectively, thus, reducing side effects.     

Synthesis and application of a new bisphosphite ligand collection for asymmetric hydroformylation of allyl cyanide

A series of mono- and bidentate phosphites was prepared with (S)-5,5',6,6'-tetramethyl-3,3'-di-tert-butyl-1,1'-biphenyl-2,2'-dioxy [(S)-BIPHEN] as a chiral auxiliary and screened in the asymmetric hydroformylation of allyl cyanide. These hydroformylation results were compared with those of two existing chiral ligands, Chiraphite and BINAPHOS, whose utility in asymmetric hydroformylation has been previously demonstrated. Bisphosphite 11 with a 2,2'-biphenol bridge was found to be the best overall ligand for asymmetric hydroformylation of allyl cyanide with up to 80% ee and regioselectivities (branch-to-linear ratio, b/l) of 20 with turnover frequency of 625 [h(-)(1)] at 35 degrees C. BINAPHOS gave enantioselectivities up to 77% ee when the reaction was conducted in either acetone or neat but with poor regioselectivity (b/l 2.8) and activities 7 times lower than that of 11. The product of allyl cyanide hydroformylation using (R,R)-11 was subsequently transformed into (R)-2-methyl-4-aminobutanol, a useful chiral building block. Single-crystal X-ray structures of (S,S)-11 and its rhodium complex 19 were determined.     

Synthesis and structural studies of some fluorescent group XII metal complexes with a terpyridine based ligand

Three 4′-(2-thienyl) substituted terpyridine (thioterpy) complexes of cadmium(II) and mercury(II) have been synthesized and fully characterized by spectroscopies. According to the crystallographic studies, the packing of these complexes was stabilized by several intermolecular interactions such as hydrogen bonding, C-H?π and π?π. Finally, the florescent properties of the prepared complexes have been investigated.