Synthesis of (S)-2-fluoro-L-daunosamine and (S)-2-fluoro-D-ristosamine

Derivatives of (S)-2-fluoro-L-daunosamine and (S)-2-fluoro-D-ristosamine were synthesized, starting ultimately from 2-amino-2-deoxy-D-glucose which was converted, according to the literature, into methyl 2-benzamido-4, 6-O-benzylidene-2-deoxy-3-O-(methylsulfonyl)-alpha-D-glucopyranoside (2). Treatment of 2 with tetrabutylammonium fluoride gave a 63% yield of (known) methyl 3-benzamido-4,6-O-benzylidene-2,3-dideoxy-2-fluoro-alpha-D-altropyran oside (4), together with a 6% yield of its 2-benzamido-2,3-dideoxy-3-fluoro-alpha-D-gluco isomer. From 4, the corresponding 6-bromo-2,3,6-trideoxyglycoside 4-benzoate (6) was obtained by Hanessian-Hullar reaction. Dehydrobromination of 6, followed by catalytic hydrogenation of the resulting 5-enoside, and subsequent debenzoylation and N-trifluoroacetylation, afforded the fluorodaunosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-beta-L-galactopyranos ide. Reductive debromination of 6, followed by debenzoylation and N-trifluoroacetylation, gave the fluororistosaminide, methyl 2,3,6-trideoxy-2-fluoro-3-trifluoroacetamido-alpha-D-altropyran oside. The 1H-n.m.r. spectra of the new aminofluoro sugars are discussed with respect to the effects of neighboring amino and acylamido substituents on geminal and vicinal 1H-19F coupling constants, in comparison with the reported effects of oxygen substituents.     

An improved protocol for the preparation of (S)-vinylglycine from (S)-methionine

We present an optimized procedure for the synthesis of (S)-vinylglycine from (S)-methionine. The key step is a solvent free pyrolysis of an intermediate sulfoxide at high temperature. Using our optimized reaction conditions, Cbz-protected vinylglycine was obtained in high yield and with almost no side products. The protocol is scalable, fast and avoids the use of poisonous reagents.     

Chiral phosphorothioate analogues of B-DNA. The crystal structure of Rp-d[Gp(S)CpGp(S)CpGp(S)C]

The compound Rp-d[Gp(S)CpGp(S)CpGp(S)C], an analogue of the deoxyoligomer d(G-C)3, crystallizes in space group P2(1)2(1)2(1) with a = 34.90 A, b = 39.15 A and c = 20.64 A. The structure, which is not isomorphous with any previously determined deoxyoligonucleotide, was refined to an R factor of 14.5% at a resolution of 2.17 A, with 72 solvent molecules located. The two strands of the asymmetric unit form a right-handed double helix, which is a new example of a B-DNA conformation and brings to light an important and overlooked component of flexibility of the double helix. This flexibility is manifest in the alternation of the backbone conformation between two states, defined by the adjacent torsion angles epsilon and zeta, trans . gauche-(BI) and gauche-. trans (BII). BI is characteristic of classical of B-DNA and has an average C(1') to C(1') separation of 4.5 A. The corresponding separation for BII is 5.3 A. Each state is associated with a distinct phosphate orientation where the plane of the PO2 (or POS) group is alternately near horizontal or vertical with respect to the helix axis. The BI and BII conformations are out of phase on the two strands. As a consequence, on one strand purine-pyrimidine stacking is better than pyrimidine-purine, while the converse holds for the other strand. At each base-pair step, good and bad stacking alternate across the helix axis. The pattern of alternation is regular in the context of a fundamental dinucleotide repeat. Re-examination of the B-DNA dodecamer d(C-G-C-G-A-A-T-T-C-G-C-G) shows that the C-G-C-G regions contain the BI and BII conformations, and the associated dual phosphate orientation and asymmetric base stacking. Different mechanisms are used in the two structures to avoid clashes between guanine residues on opposite strands, a combination of lateral slide, tilt and helical twist in the present structure, and base roll, tilt and longitudinal slide (Calladine rules) in the dodecamer. The flexibility of the phosphate orientations demonstrated in this structure is important, since it offers a structural basis for protein-nucleic acid recognition.     

Asymmetric aldol reaction of enol trichloroacetate catalyzed by (S, S)-(EBTHI)TiCl(OMe)

(S,S)-Ethylenebis(tetrahydroindenyl)titanium chloride methoxide, (S, S)-(EBTHI)TiCl(OMe) (3) was synthesized from the corresponding titanium dichloride. The asymmetric aldol reaction of enol trichloroacetate of cyclohexanone 1 with aromatic aldehydes was studied in the presence of a catalytic amount of the chiral titanium complex 3, with the result that the optically active syn aldol adduct 2 was preferentially obtained with up to 91% ee.     

Synthesis and antioxidant evaluation of (S,S)- and (R,R)-secoisolariciresinol diglucosides (SDGs)

Secoisolariciresinol diglucosides (SDGs) (S,S)-SDG-1 (major isomer in flaxseed) and (R,R)-SDG-2 (minor isomer in flaxseed) were synthesized from vanillin via secoisolariciresinol (6) and glucosyl donor 7 through a concise route that involved chromatographic separation of diastereomeric diglucoside derivatives (S,S)-8 and (R,R)-9. Synthetic (S,S)-SDG-1 and (R,R)-SDG-2 exhibited potent antioxidant properties (EC₅₀ = 292.17 ± 27.71 μM and 331.94 ± 21.21 μM, respectively), which compared well with that of natural (S,S)-SDG-1 (EC₅₀ = 275.24 ± 13.15 μM). These values are significantly lower than those of ascorbic acid (EC₅₀ = 1129.32 ± 88.79 μM) and α-tocopherol (EC₅₀ = 944.62 ± 148.00 μM). Compounds (S,S)-SDG-1 and (R,R)-SDG-2 also demonstrated powerful scavenging activities against hydroxyl [natural (S,S)-SDG-1: 3.68 ± 0.27; synthetic (S,S)-SDG-1: 2.09 ± 0.16; synthetic (R,R)-SDG-2: 1.96 ± 0.27], peroxyl [natural (S,S)-SDG-1: 2.55 ± 0.11; synthetic (S,S)-SDG-1: 2.20 ± 0.10; synthetic (R,R)-SDG-2: 3.03 ± 0.04] and DPPH [natural (S,S)-SDG-1: EC₅₀ = 83.94 ± 2.80 μM; synthetic (S,S)-SDG-1: EC₅₀ = 157.54 ± 21.30 μM; synthetic (R,R)-SDG-2: EC₅₀ = 123.63 ± 8.67 μM] radicals. These results confirm previous studies with naturally occurring (S,S)-SDG-1 and establish both (S,S)-SDG-1 and (R,R)-SDG-2 as potent antioxidants and free radical scavengers for potential in vivo use.     

Rabbit reticulocyte lipoxygenase catalyzes specific 12(S) and 15(S) oxygenation of arachidonoyl-phosphatidylcholine

The rabbit reticulocyte lipoxygenase is known to display an unusual facility for oxygenation of esterified polyunsaturated fatty acids, yet the precise structures of the products are not known. With free arachidonate as substrate the enzyme is known to catalyze 15S and 12S oxygenations, and demonstration of a facility for catalysis of these reactions on phospholipids would extend the potential scope of lipoxygenase reactions in cells. We elected to study in detail the reaction of the enzyme with a natural phospholipid, palmitoyl/arachidonoyl-phosphatidylcholine. We determined the nature of the products by initial isolation by RP-HPLC, followed by transesterification and identification of the oxygenated products by HPLC, uv, GC-MS, and steric analysis of hydroxyl configuration by HPLC. The major product was identified as a phosphatidylcholine in which the arachidonate component was converted to the 15(S)-hydroperoxy-eicosatetraenoate. A second oxygenated phospholipid was produced in smaller quantities (2-5% of the latter product) and identified as the 12(S)-oxygenated analog. These products were also identified after reaction of the reticulocyte lipoxygenase with human red cell membranes which were radiolabeled by preincubation with [3H]arachidonic acid. The finding of 12S oxygenation represents the first evidence that a lipoxygenase can control a reaction centered on the 10-carbon of an arachidonoyl phospholipid. This is an important precedent, because hydrogen abstraction from carbon-10 is a critical step in the lipoxygenase-catalyzed synthesis of 8- and 12-hydroperoxy-eicosatetraenoates (HPETEs) and for the conversion of 5- and 15-HPETEs to leukotrienes.     

Resin effects in solid phase S(n)Ar and S(n)2 macrocyclizations

Seven different supports were compared in solid-phase S(N)Ar and S(N)2 macrocyclization reactions. Product purities were assayed for a relatively facile ring-closure process to give products 1 and 3. Some less-facile ring-closure reactions give the undesired dimeric macrocyclization by-products 2; some of these more-demanding ring closures were also examined. Finally, experiments were performed to gauge the rate of cyclizations on different resins, and some qualitative data were obtained for this.     

Production of 10(S)-hydroxy-8(E)-octadecenoic and 7,10(S,S)-hydroxy-8(E)-octadecenoic ethyl esters by Novozym 435 in solvent-free media

Novozym 435, lipase B from Candida antarctica, was used in this study for the production of ethyl esters. For the first time, trans-hydroxy-fatty acid ethyl esters were synthesized in vitro in solvent-free media. We studied the effects of the substrate–ethanol molar ratio and enzyme synthetic stability of the biocatalyst. To determine the structure of the formed compounds, Fourier transformed infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry were used, three less time-consuming structural techniques. trans-Hydroxy-fatty acid ethyl esters were synthesized with a reaction yield of 90 % or higher with optimal reaction conditions.     

Model-free analysis of a thermophilic Fe(7)S(8) protein compared with a mesophilic Fe(4)S(4) protein

15N T(1), T(2) and (1)H-(15)N NOE were measured for the thermophilic Fe(7)S(8) protein from Bacillus schlegelii and for the Fe(4)S(4) HiPIP protein from Chromatium vinosum, which is a mesophilic protein. The investigation was performed at 276, 300, and 330 K at 11.7 T for the former, whereas only the 298 K data at 14.1 T for the latter were acquired. The data were analyzed with the model-free protocol after correcting the measured parameters for the effect of paramagnetism, because both proteins are paramagnetic. Both thermophilic and mesophilic proteins are quite rigid, with an average value of the generalized order parameter S2at room temperature of 0.92 and 0.94 for Fe(7)S(8) and Fe(4)S(4) proteins, respectively. The analyzed nitrogens for the Fe(7)S(8) protein showed a significant decrease in S2with increasing temperature, and at the highest temperature >70% of the residues had an internal correlation time. This research shows that subnanosecond rigidity is not related to thermostability and provides an estimate of the effect of increasing temperature on this time scale.     

Regulation of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding sites on human epidermal cells by interferon-gamma

We recently detected specific high-affinity binding sites for 12(S)-HETE, the main arachidonic acid metabolite in skin, on epidermal cells. The putative receptor is involved in keratinocyte chemotaxis toward 12(S)-HETE, which points to its participation in wound healing. In an effort to further characterize the 12(S)-HETE receptor, we investigated its regulation by various cytokines. Of the tested cytokines, only interferon (IFN)-gamma led to a massive induction of the 12(S)-HETE receptors. The effect was dose and time dependent and blocked by cycloheximide. The up-regulation of 12(S)-HETE receptors by IFN-gamma may represent an amplification mechanism of the assumed role of 12(S)-HETE in skin wound repair.     

beta-cyclodextrin-immobilized (4S)-phenoxy-(S)-proline as a catalyst for direct asymmetric aldol reactions

beta-Cyclodextrin-immobilized (4S)-phenoxy-(S)-proline was prepared conveniently by simply heating the amino acid and beta-cyclodextrin in ethanol-water (1/1, v/v) and removal of the solvent. This proved to be an efficient catalyst for direct asymmetric aldol reactions, and the catalyst could be recycled four times without loss of enantioselectivity.     

High-affinity binding sites for 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) in carcinoma cells.

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE) enhances tumor cell adhesion to endothelial cells [Honn et al. (1988) Proc. Soc. Exp. Biol. Med. 189, 130-135]. The effect is correlated to surface expression of an integrin receptor, GpIIb/IIIa. Here, we describe evidence for high-affinity binding of 12(S)-HETE to Lewis lung carcinoma cells. Scatchard plot analyses indicated a single class of sites with apparent Kd and Bmax values of 0.44 nM and 66,000 sites per cell, respectively. Competition experiments with unlabeled compounds shod d that the binding was reversible and saturable as well as stereo- and regiospecific. The 12(S)-HETE binding, demonstrated here, might be an important step in a series of events controlling surface expression of integrin receptors.     

PLP-dependent H(2)S biogenesis

The role of endogenously produced H(2)S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H(2)S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H(2)S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5'-phosphate-dependent (PLP) enzymes, cystathionine β-synthase and cystathionine γ-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H(2)S can be liberated. In this review, we present an overview of the kinetics of the H(2)S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.     

Synthesis of (R)- and (S)- muscone

(R)-(-)-Muscone (3-methylcyclopentadecanone, 1) the key perfumery component isolated from the male musk deer, Moschus moschiferus,* was synthesized from the easily available chiral building block, (R)-3-tert-butoxycarbonyl-2-methylpropanoic acid (2), by employing ring-closing olefin metathesis (RCM). Antipode (+)-1 was also synthesized in a similar manner from tert-butyl (S)-3-methoxycarbonylbutanoate (10). *(a) Walbaum, H. J. J. Prakt. Chem., 73, 488 (1906); (b) Ruzicka, L., Further considerations on the constitution of muscone. Helv. Chim. Acta, 9, 715, 1008-1017 (1926).