Synthesis of (-)-dactylolide and 13-desmethylene-(-)-dactylolide and their effects on tubulin

An efficient new synthesis has been elaborated for non-natural (-)-dactylolide ((-)-2) and its 13-desmethylene analogue 4, employing a HWE-based macrocyclization approach with beta-keto-phosphonate/aldehyde 19 and the respective 13-desmethylene derivative as the key intermediates. Both (-)-2 and 4 as well as the corresponding C20 alcohols inhibit human cancer cell proliferation with IC(50) values in the sub-micromolar range and induce the polymerization of tubulin in vitro.

Intermolecular Clamping by Hydrogen Bonds: 2-Pyridone center dot NH3

A combined spectroscopic and ab initio theoretical study of the doubly hydrogen-bonded complex of 2-pyridone (2PY) with NH3 has been performed. The S-1 <- S-0 spectrum extends up to approximate to 1200 cm(-1) above the 0(0)(0) band, close to twice the range observed for 2PY. The S-1 state nonradiative decay for vibrations above approximate to 300 cm(-1) in the NH3 complex is dramatically slowed down relative to bare 2PY. Also, the Delta v=2,4,... overtone bands of the v(1)' and v(2)' out-of-plane vibrations that dominate the low-energy spectral region of 2PY are much weaker or missing for 2PY center dot NH3, which implies that the bridging (2PY)NH center dot center dot center dot NH3 and H2NH center dot center dot center dot O=C H-bonds clamp the 2PY at a planar geometry in the S-1 state. The mass-resolved UV vibronic spectra of jet-cooled 2PY center dot NH3 and its H/D mixed isotopomers are measured using two-color resonant two-photon ionization spectroscopy. The S-0 and S-1 equilibrium structures and normal-mode frequencies are calculated by density functional (B3LYP) and correlated ab initio methods (MP2 and approximate second-order coupled-cluster, CC2). The S-1 <- S-0 vibronic assignments are based on configuration interaction singles (CIS) and CC2 calculations. A doubly H-bonded bridged structure of C-S symmetry is predicted, in agreement with that of Held and Pratt [J. Am. Chem. Soc. 1993, 115, 9718]. While the B3LYP and MP2 calculated rotational constants are in very good agreement with experiment, the calculated H2NH center dot center dot center dot O=C H-bond distance is approximate to 0.7 angstrom shorter than that derived by Held and Pratt. On the other hand, this underlines their observation that ammonia can act as a strong H-bond donor when built into an H-bonded bridge. The CC2 calculations predict the H2NH center dot center dot center dot O distance to increase by 0.2 angstrom upon S-1 <- S-0 electronic excitation, while the (2PY)NH center dot center dot center dot NH3 H-bond remains nearly unchanged. Thus, the expansion of the doubly H-bonded bridge in the excited state is asymmetric and almost wholly due to the weakening of the interaction of ammonia with the keto acceptor group.

S-0 and S-1 State Structure, Methyl Torsional Barrier Heights, and Fast Intersystem Crossing Dynamics of 5-Methyl-2-hydroxypyrimidine

We report the analysis of the SI So rotational band contours of jet-cooled 5-methyl-2-hydroxypyrimidine (5M2HP), the enol form of deoxythymine. Unlike thymine, which exhibits a structureless spectrum, the vibronic spectrum of 5M2HP is well structured, allowing us to determine the rotational constants and the methyl group torsional barriers in the S-0 and S-1 states. The 0(0)(0), 6a(0)(1), 6b(0)(1), and 14(0)(1) band contours were measured at 900 MHz (0.03 cm(-1)) resolution using mass-specific two-color resonant two-photon ionization (2C-R2PI) spectroscopy. All four bands are polarized perpendicular to the pyrimidine plane (>90% c type), identifying the S-1 <- S-0 excitation of 5M2HP as a 1n pi* transition. All contours exhibit two methyl rotor subbands that arise from the lowest 5-methyl torsional states 0A '' and 1E ''. The S-0 and S-1 state torsional barriers were extracted from fits to the torsional subbands. The 3-fold barriers are V-3 '' = 13 cm(-1) and V3' = SI cm(-1); the 6-fold barrier contributions V-6 '' and V-6' are in the range of 2-3 cm(-1) and are positive in both states. The changes of A, B, and C rotational constants upon S-1 <- S-0 excitation were extracted from the contours and reflect an "anti-quinoidal" distortion. The 0(0)(0) contour can only be simulated if a 3 GHz Lorentzian line shape is included, which implies that the S-1(1n pi*) lifetime is similar to 55 ps. For the 6a(0)(1) and 6b(0)(1) bands, the Lorentzian component increases to 5.5 GHz, reflecting a lifetime decrease to similar to 30 ps. The short lifetimes are consistent with the absence of fluorescence from the 1n pi* state. Combining these measurements with the previous observation of efficient intersystem crossing (ISC) from the Si state to a long-lived T-1((3)n pi*) state that lies similar to 2200 cm(-1) below [S. Lobsiger, S. et al. Phys. Chem. Chem. Phys. 2010, 12, 5032] implies that the broadening arises from fast intersystem crossing with k(ISC) approximate to 2 x 10(10) s(-1). In comparison to 5-methylpyrimidine, the ISC rate is enhanced by at least 10 000 by the additional hydroxy group in position 2.

Influence of GABA(A) receptor α subunit isoforms on the benzodiazepine binding site

Classical benzodiazepines, such as diazepam, interact with α(x)β(2)γ(2) GABA(A) receptors, x = 1, 2, 3, 5 and modulate their function. Modulation of different receptor isoforms probably results in selective behavioural effects as sedation and anxiolysis. Knowledge of differences in the structure of the binding pocket in different receptor isoforms is of interest for the generation of isoform-specific ligands. We studied here the interaction of the covalently reacting diazepam analogue 3-NCS with α(1)S204Cβ(2)γ(2), α(1)S205Cβ(2)γ(2) and α(1)T206Cβ(2)γ(2) and with receptors containing the homologous mutations in α(2)β(2)γ(2), α(3)β(2)γ(2), α(5)β(1/2)γ(2) and α(6)β(2)γ(2). The interaction was studied using radioactive ligand binding and at the functional level using electrophysiological techniques. Both strategies gave overlapping results. Our data allow conclusions about the relative apposition of α(1)S204Cβ(2)γ(2), α(1)S205Cβ(2)γ(2) and α(1)T206Cβ(2)γ(2) and homologous positions in α(2), α(3), α(5) and α(6) with C-atom adjacent to the keto-group in diazepam. Together with similar data on the C-atom carrying Cl in diazepam, they indicate that the architecture of the binding site for benzodiazepines differs in each GABA(A) receptor isoform α(1)β(2)γ(2), α(2)β(2)γ(2), α(3)β(2)γ(2), α(5)β(1/2)γ(2) and α(6)β(2)γ(2).

Total synthesis of (-)-zampanolide and structure-activity relationship studies on (-)-dactylolide derivatives

A new total synthesis of the marine macrolide (-)-zampanolide (1) and the structurally and stereochemically related non-natural levorotatory enantiomer of (+)-dactylolide (2), that is, ent-2, has been developed. The synthesis features a high-yielding, selective intramolecular Horner-Wadsworth-Emmons (HWE) reaction to close the 20-membered macrolactone ring of 1 and ent-2. The β-keto phosphonate/aldehyde precursor for the ring-closure reaction was obtained by esterification of a ω-diethylphosphono carboxylic acid fragment and a secondary alcohol fragment incorporating the THP ring that is embedded in the macrocyclic core structure of 1 and ent-2. THP ring formation was accomplished through a segment coupling Prins-type cyclization. Employing the same overall strategy, 13-desmethylene-ent-2 as well as the monocyclic desTHP derivatives of 1 and ent-2 were prepared. Synthetic 1 inhibited human cancer cell growth in vitro with nM IC(50) values, while ent-2, which lacks the diene-containing hemiaminal-linked side chain of 1, is 25- to 260-fold less active. 13-Desmethylene-ent-2 as well as the reduced versions of ent-2 and 13-desmethylene-ent-2 all showed similar cellular activity as ent-2 itself. The same activity level was attained by the monocyclic desTHP derivative of 1. Oxidation of the aldehyde functionality of ent-2 gave a carboxylic acid that was converted into the corresponding N-hexyl amide. The latter showed only μM antiproliferative activity, thus being several hundred-fold less potent than 1.

Capillary electrophoresis contributions to the hydromorphone metabolism in man

CE-ESI multistage IT-MS (CE-MS(n), n < or = 4) and computer simulation of fragmentation are demonstrated to be effective tools to detect and identify phase I and phase II metabolites of hydromorphone (HMOR) in human urine. Using the same CE conditions as previously developed for the analysis of urinary oxycodone and its metabolites, HMOR and its phase I metabolites produced by N-demethylation, 6-keto-reduction and N-oxidation and phase II conjugates of HMOR and its metabolites formed with glucuronic acid, glucose, and sulfuric acid could be detected in urine samples of a patient that were collected during a pharmacotherapy episode with daily ingestion of 48 mg of HMOR chloride. The CE-MS(n) data obtained with the HMOR standard, synthesized hydromorphol and hydromorphone-N-oxide, and CYP3A4 in vitro produced norhydromorphone were employed to identify the metabolites. This approach led to the identification of previously unknown HMOR metabolites, including HMOR-3O-glucide and various N-oxides, structures for which no standard compounds or mass spectra library data were available. Furthermore, the separation of alpha- and beta-hydromorphol, the stereoisomers of 6-keto-reduced HMOR, was achieved by CE in the presence of the single isomer heptakis(2,3-diacetyl-6-sulfato)-beta-CD. The obtained data indicate that the urinary excretion of alpha-hydromorphol is larger than that of beta-hydromorphol.

Molecular interactions underlying the unusually high adenosine affinity of a novel Trypanosoma brucei nucleoside transporter

Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (Km = 0.068 +/- 0.013 microM), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and long-slender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3'-hydroxy, 5'-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (DeltaG0 = 12 kJ/mol), explaining the different Km values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.

Enzyme engineering of aminotransferases for improved activity and thermostability

The production by biosynthesis of optically active amino acids and amines satisfies the pharmaceutical industry in its demand for chiral building blocks for the synthesis of various pharmaceuticals. Among several enzymatic methods that allow the synthesis of optically active aminoacids and amines, the use of minotransferase is a promising one due to its broad substrate specificity and no requirement for external cofactor regeneration. The synthesis of chiral compounds by aminotransferases can be done either by asymmetric synthesis starting from keto acids or ketones, and by kinetic resolution starting from racemic aminoacids or amines. The asymmetric synthesis of substituted (S)-aminotetralin, an active pharmaceutical ingredient (API), has shown to have two major factors that contribute to increasing the cost of production. These factors are the raw material cost of biocatalyst used to produce it and product loss during biocatalyst separation. To minimize the cost contribution of biocatalyst and to minimize the loss of product, two routes have been chosen in this research: 1. To engineer the aminotransferase biocatalyst to have greater specific activity, and 2. Improve the engineering of the process by immobilization of biocatalyst in calcium alginate and addition of cosolvents. An (S)-aminotransferase (Mutant CNB03-03) was immobilized, not as purified enzyme but as enzyme within spray dried cells, in calcium alginate beads and used to produce substituted (S)-aminotetralin at 50 °C and pH 7 in experiments where the immobilized biocatalyst was recycled. Initial rate of reaction for cycle 1 (6 hr duration) was determined to be 0.258 mM/min, for cycle 2 (20 hr duration) it decreased by ~50% compared to cycle 1, and for cycle 3 (20 hr duration) it decreased by ~90% compared to cycle 1 (immobilized preparation consisted of 50 mg of spray dried cells per gram of calcium alginate). Conversion to product for each cycle decreased as well, from 100% in cycle 1 (About 50 mM), 80% in cycle 2, and 30% after cycle 3. This mutant was determined to be deactivated at elevated temperatures during the reaction cycle and was not stable enough to allow multiple cycles in its immobilized form. A new mutant aminotransferase was isolated by applying error-prone polymerase chain reaction (PCR) on the gene coding for this enzyme and screening/selection: CNB04-01. This mutant showed a significant improvement in thermostability in comparison to CNB03-03. The new mutant was immobilized and tested under similar reaction conditions. Initial rate remained fairly constant (0.2 mM/min) over four cycles (each cycle with a duration of about 20 hours) with the mutant retaining almost 80% of initial rate in the fourth cycle. The final product concentrations after each cycle did not decrease during recycle experiments. Thermostability of CNB04-01 was much improved compared to CNB03-03. Under the same reaction conditions as stated above, the addition of co-solvents was studied in order to increase substituted tetralone solubility. Toluene and sodium dodecylsulfate (SDS) were used. SDS at 0.01% (w/v) allowed four recycles of the immobilized spray dried cells of CNB04-01, always reaching higher product concentration (80-85 mM) than the system with toluene at 3% (v/v) -70 mM-. The long term activity of immobilized CNB04-01 in a system with SDS 0.01% (w/v) at 50 °C, pH 7 was retained for three cycles (20 to 24 hours each one), reaching always final product concentration between 80-85 mM, but dropping precipitously in the fourth cycle to a final product concentration of 50 mM. Although significant improvement of immobilization on productivity and stability were observed using CNB04-01, another observation demonstrated the limitations of an immobilization strategy on reducing process costs. After analyzing the results of this experiment it was seen that a sudden drop occurred on final product concentration after the third recycle. This was due to product accumulation inside the immobilized preparation. In order to improve the economics of the process, research was focused on developing a free enzyme with an even higher activity, thus reducing raw material cost as well as improving biomass separation. A new enzyme was obtained (CNB05-01) using error-prone PCR and screening using as a template the gene derived from the previous improved enzyme. This mutant was determined to have 1.6 times the initial rate of CNB04-01 and had a higher temperature optimum (55°). This new enzyme would allow reducing enzyme loading in the reaction by five-fold compared to CNB03-03, when using it at concentration of one gram of spray dried cells per liter (completing the reaction after 20-24 hours). Also this mutant would allow reducing process time to 7-8 hours when used at a concentration of 5 grams of spray dried cells per liter compared to 24 hours for CNB03-03, assuming that the observations shown before are scalable. It could be possible to improve the economics of the process by either reducing enzyme concentration or reducing process time, since the production cost of the desired product is primarily a function of both enzyme concentration and process time.

Convenient synthesis of substituted α-methylene--valerolactones in aqueous medium using Baylis-Hillman chemistry

A mild and convenient synthesis of substituted α-methylene--valerolactones was achieved by SN2 nucleophilic substitution of the acetates of the Baylis-Hillman adducts with acetyl acetone followed by one-pot saponification of the ester, reduction of the keto group and subsequent intramolecular ring closure in aqueous medium.

A general approach to the synthesis of substituted isoxazolo[4,3-c]quinolines via chalcones

A general and practical approach to the synthesis of substituted isoxazolo[4,3-c]quinolines from the substituted isoxazolines afforded by 1,3-dipolar cycloaddition between 2-nitrobenzonitrile oxide and chalcones is described. The SnCl2.2H2O-mediated reduction of the nitro group followed by intramolecular cyclization involving the amino and the keto groups in these substrates furnished a mixture of isoxazolo[4,3- c]-quinolines and 3,5-dihydro-isoxazolo[4,3-c]quinoline. In contrast, the reduction of these substrates with Fe-AcOH unexpectedly yielded 3-benzoyl-4-quinolinamine derivatives.

Synthesis and Biological Activity of 7,8,9-Trideoxy- and 7 R DesTHP-Peloruside A

The stereoselective syntheses of 7,8,9-trideoxypeloruside A (4) and a monocyclic peloruside A analogue lacking the entire tetrahydropyran moiety (3) are described. The syntheses proceeded through the PMB-ether of an ω-hydroxy β-keto aldehyde as a common intermediate which was elaborated into a pair of diastereomeric 1,3-syn and -anti diols by stereoselective Duthaler–Hafner allylations and subsequent 1,3-syn or anti reduction. One of these isomers was further converted into a tetrahydropyran derivative in a high-yielding Prins reaction, to provide the precursor for bicyclic analogue 4. Downstream steps for both syntheses included the substrate-controlled addition of a vinyl lithium intermediate to an aldehyde, thus connecting the peloruside side chain to C15 (C13) of the macrocyclic core structure in a fully stereoselective fashion. In the case of monocyclic 3 macrocyclization was based on ring-closing olefin metathesis (RCM), while bicyclic 4 was cyclized through Yamaguchi-type macrolactonization. The macrolactonization step was surprisingly difficult and was accompanied by extensive cyclic dimer formation. Peloruside A analogues 3 and 4 inhibited the proliferation of human cancer cell lines in vitro with micromolar and sub-micromolar IC50 values, respectively. The higher potency of 4 highlights the importance of the bicyclic core structure of peloruside A for nM biological activity.

Watson–Crick and Sugar-Edge Base Pairing of Cytosine in the Gas Phase: UV and Infrared Spectra of Cytosine·2-Pyridone

While keto-amino cytosine is the dominant species in aqueous solution, spectroscopic studies in molecular beams and in noble gas matrices show that other cytosine tautomers prevail in apolar environments. Each of these offers two or three H-bonding sites (Watson–Crick, wobble, sugar-edge). The mass- and isomer-specific S1 ← S0 vibronic spectra of cytosine·2-pyridone (Cyt·2PY) and 1-methylcytosine·2PY are measured using UV laser resonant two-photon ionization (R2PI), UV/UV depletion, and IR depletion spectroscopy. The UV spectra of the Watson–Crick and sugar-edge isomers of Cyt·2PY are separated using UV/UV spectral hole-burning. Five different isomers of Cyt·2PY are observed in a supersonic beam. We show that the Watson–Crick and sugar-edge dimers of keto-amino cytosine with 2PY are the most abundant in the beam, although keto-amino-cytosine is only the third most abundant tautomer in the gas phase. We identify the different isomers by combining three different diagnostic tools: (1) methylation of the cytosine N1–H group prevents formation of both the sugar-edge and wobble isomers and gives the Watson–Crick isomer exclusively. (2) The calculated ground state binding and dissociation energies, relative gas-phase abundances, excitation and the ionization energies are in agreement with the assignment of the dominant Cyt·2PY isomers to the Watson–Crick and sugar-edge complexes of keto-amino cytosine. (3) The comparison of calculated ground state vibrational frequencies to the experimental IR spectra in the carbonyl stretch and NH/OH/CH stretch ranges strengthen this identification.

Excited-State Structure, Vibrations, and Nonradiative Relaxation of Jet-Cooled 5-Fluorocytosine

The S0 → S1 vibronic spectrum and S1 state nonradiative relaxation of jet-cooled keto-amino 5-fluorocytosine (5FCyt) are investigated by two-color resonant two-photon ionization spectroscopy at 0.3 and 0.05 cm–1 resolution. The 000 rotational band contour is polarized in-plane, implying that the electronic transition is 1ππ*. The electronic transition dipole moment orientation and the changes of rotational constants agree closely with the SCS-CC2 calculated values for the 1ππ* (S1) transition of 5FCyt. The spectral region from 0 to 300 cm–1 is dominated by overtone and combination bands of the out-of-plane ν1′ (boat), ν2′ (butterfly), and ν3′ (HN–C6H twist) vibrations, implying that the pyrimidinone frame is distorted out-of-plane by the 1ππ* excitation, in agreement with SCS-CC2 calculations. The number of vibronic bands rises strongly around +350 cm–1; this is attributed to the 1ππ* state barrier to planarity that corresponds to the central maximum of the double-minimum out-of-plane vibrational potentials along the ν1′, ν2′, and ν3′ coordinates, which gives rise to a high density of vibronic excitations. At +1200 cm–1, rapid nonradiative relaxation (knr ≥ 1012 s–1) sets in, which we interpret as the height of the 1ππ* state barrier in front of the lowest S1/S0 conical intersection. This barrier in 5FCyt is 3 times higher than that in cytosine. The lifetimes of the ν′ = 0, 2ν1′, 2ν2′, 2ν1′ + 2ν2′, 4ν2′, and 2ν1′ + 4ν2′ levels are determined from Lorentzian widths fitted to the rotational band contours and are τ ≥ 75 ps for ν′ = 0, decreasing to τ ≥ 55 ps at the 2ν1′ + 4ν2′ level at +234 cm–1. These gas-phase lifetimes are twice those of S1 state cytosine and 10–100 times those of the other canonical nucleobases in the gas phase. On the other hand, the 5FCyt gas-phase lifetime is close to the 73 ps lifetime in room-temperature solvents. This lack of dependence on temperature and on the surrounding medium implies that the 5FCyt nonradiative relaxation from its S1 (1ππ*) state is essentially controlled by the same ∼1200 cm–1 barrier and conical intersection both in the gas phase and in solution.

Synthesis and incorporation into a-DNA of a novel conformationally constrained alpha-nucleoside analogue

We describe the synthesis and incorporation into alpha-DNA of a novel conformationally constrained alpha-nucleoside analogue. The carbohydrate part of this analogue was prepared in 4 steps from the known bicyclic precursor 1 via a stereospecific, intramolecular, Et 3B mediated radical addition to a keto-function as the key step. The thus obtained intermediate 4 was transformed stereoselectively into the corresponding alpha-nucleoside analogues 7 and 8 containing the bases adenine and thymine, and were further elaborated into the phosphoramidite building blocks 11 and 12 . Both building blocks were incorporated into alpha-oligodeoxynucleotides and their pairing behavior to parallel complementary DNA studied by UV-melting experiments. Single substitutions of alpha-deoxyribnucleoside units by the new analogues in the center of duplexes were found to be thermally destabilizing by only -0.8 to -3.1›C.

Heterocyst glycolipids in surface sediments and water samples of the Amazon shelf

Marine endosymbiotic heterocystous cyanobacteria make unique heterocyst glycolipids (HGs) containing pentose (C5) moieties. Functionally similar HGs with hexose (C6) moieties found in free-living cyanobacteria occur in the sedimentary record, but C5 HGs have not been documented in the natural environment. Here we developed a high performance liquid chromatography multiple reaction monitoring (MRM) mass spectrometry (HPLC-MS2) method specific for trace analysis of long chain C5HGs and applied it to cultures of Rhizosolenia clevei Ostenfeld and its symbiont Richelia intracellularis which were found to contain C5 HGs and no C6 HGs. The method was then applied to suspended particulate matter (SPM) and surface sediment from the Amazon plume region known to harbor marine diatoms carrying heterocystous cyanobacteria as endosymbionts. C5 HGs were detected in both marine SPM and surface sediments, but not in SPM or surface sediment from freshwater settings in the Amazon basin. Rather, the latter contained C6 HGs, established biomarkers for free-living heterocystous cyanobacteria. Our results indicate that the C5 HGs may be potential biomarkers for marine endosymbiotic heterocystous cyanobacteria.