A novel class of endothelin-A receptor antagonists, (R)-2-(benzo[1,3]dioxol-5-yl)-6-isopropyloxy-2H-chromene-3-carboxylic acids (S-1255). Conformational analysis of basic structure,crucial for ET(A) antagonism,in solution and solid states

Conformational studies of potent and selective endothelin-A (ET(A)) receptor antagonists, 4-substituted (R)-2-(benzo[1,3]-dioxol-5-yl)-6-isopropoxy-2H-chromene-3-carboxylic acids, are reported. X-ray crystallography and NMR studies of the 4-anisyl derivative 2 (S-1255), the stable atropisomers 3 and the 4-n-butyl derivative 4 reveal that the A-, B- and C-rings in these compounds adopt a L-like conformation in both solution and solid states. Molecular mechanics calculation shows that this L-like conformation is an inevitable conformation as determined by intramolecular steric repulsions. These 2H-chromene derivatives bound to an ET(A) receptor with IC(50) values of less than 1 nM, whereas the dihydro compounds 7 and 9 not having the L-like conformation showed weaker affinities. These results suggest that the L-like conformation is specifically recognized by the active site of the ET(A) receptor. The roles of the L-like conformation in the receptor binding are discussed.     

Isomerisation and conformation studies of (+)- and (-)6-hydroxy-5, 6-dihydrouridine.

(1) "Uridine hydrates" i.e. (+)- and (-)6-hydroxy-5, 6-dihydrouridine were formed under gamma irradiation in a deaerated aqueous solution of uridine. (2) The structures of two diastereoisomers were determined by spectroscopic measurements (infrared, ultraviolet and NMR) and verified by stereospecific synthesis; uridine hydrates were prepared by mild reduction of trans(+)- and (-)iodohydrins with acetic acid and zinc power. (3) The carbon 6 epimerisation of uridine hydrates 6R or 6S was performed in triated water (pH 5.5, 30 degrees C) and at the same time tritium incorporation on carbon 5 was noted. The mechanism of these reactions could be explained by the opening of the N1-C6 bond of the pyrimidine ring, followed by ketoenolisation reaction of carbons 4 and 5. (4) The 250 MHz NMR analysis has allowed us to determine the nucleoside conformations. Nucleosides had mainly the S(C2' endo) conformation. A slight preference of gauche-gauche (gg) rotamer of the exocyclic hydroxymethyl group was noted and the aglycone was in the anti conformation.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Human serum albumin hybrid incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer as an O2-binding site

We have found that recombinant human serum albumin (HSA) incorporating tailed porphyrinatoiron(II) in the alpha,alpha,alpha,beta-conformer can reversibly bind and release O2 under physiological conditions (pH 7.3, 37 degrees C) like hemoglobin and myoglobin. beta-2-Methylimidazolyl-tailed porphyrinatoirons (6a, 6b) are synthesized via four steps from the atropisomers of tetrakis(o-aminophenyl)porphyrin. The stereochemistry of the alpha,alpha,alpha,beta-conformer has been determined by NMR spectroscopy. 6a and 6b form stable O2-adduct complexes in toluene solution at room temperature. The association rate constants of O2 are 3.1- and 1.9-fold lower than those of the corresponding alpha,alpha,alpha,alpha-conformers (1a, 1b), indicating that the three substituents (cyclohexanamide or pivalamide groups) are close to each other on the porphyrin platform and construct a narrow encumbrance around the O2-coordination site. Although 6a and 6b are incorporated into the hydrophobic domains of HSA to produce the albumin-heme hybrid, only HSA-6a can bind O2 in aqueous medium. The cyclohexanamide fences are necessary for the tailed porphyrinatoiron to form a stable O2-adduct complex under physiological conditions. The O2-binding affinity (P(1/2)) of HSA-6a is 45 Torr (37 degrees C), and the O2 transporting efficiency between lungs and muscle tissues in the human body is estimated to be identical to that of human red blood cells. The HSA-6a solution will become one of the most promising materials for red blood cell substitutes, which can be manufactured on an industrial scale.     

Solution properties of antitumor sulfated derivative of alpha-(1-->3)-D-glucan from Ganoderma lucidum

Four fractions of a water-insoluble alpha-(1-->3)-D-glucan GL extracted from fruiting bodies of Ganoderma lucidum were dissolved in 0.25 M LiCl/DMSO, and then reacted with sulfur trioxide-pyridine complex at 80 degrees C to synthesize a series of water-soluble sulfated derivatives S-GL. The degree of substitution of DS was measured by using IR infrared spectra, elemental analysis, and 13C NMR to be 1.2-1.6 in the non-selective sulfation. Weight-average molecular weight Mw and intrinsic viscosity [eta] of the sulfated derivatives S-GL were measured by multi-angle laser light scattering and viscometry. The Mw value (2.4 x 10(4)) of sulfated glucan S-GL-1 was much lower than that (44.5 x 10(4)) of original alpha-(1-->3)-D-glucan GL-1. The Mark-Houwink equation and average value of characteristic ratio C(infinity) for the S-GL in 0.2 M NaCl aqueous solution at 25 degrees C were found to be: [eta] = 1.32 x 10(-3) Mw(1.06) (cm3 g(-1)) and 16, respectively, in the Mw range from 1.1 x 10(4) to 2.4 x 10(4). It indicated that the sulfated derivatives of the alpha-(1-->3)-D-glucan in the aqueous solution behave as an expanded chain, owing to intramolecular hydrogen bonding or interaction between charge groups. Interestingly, two sulfated derivatives synthesized from the alpha-(1-->3)-D-glucan and curdlan, a beta-(1-->3)-D-glucan, all had significant higher antitumor activity against Ehrlich ascites carcinoma (EAC) than the originals. The effect of expanded chains of the sulfated glucan in the aqueous solution on the improvement of the antitumor activity could not be negligible.     

Intercalation of the (-)-(1R,2S,3R, 4S)-N6-[1-benz[a]anthracenyl]-2'-deoxyadenosyl adduct in an oligodeoxynucleotide containing the human N-ras codon 61 sequence

The solution structure of the (-)-(1R,2S,3R,4S)-N6-[1-(1,2,3, 4-tetrahydroxy-benz[a]anthracenyl)]-2'-deoxyadenosyl adduct at X6 of 5'-d(CGGACXAGAAG)-3'.5'-d(CTTCTTGTCCG)-3', incorporating codons 60, 61(italic), and 62 of the human N-ras protooncogene, was determined. This adduct results from the trans opening of 1S,2R,3R,4S-1, 2-epoxy-1,2,3,4-tetrahydro-benz[a]anthracenyl-3,4-diol by the exocyclic N6 of adenine. Molecular dynamics simulations were restrained by 509 NOEs from 1H NMR. The precision of the refined structures was monitored by pairwise root-mean-square deviations which were <1.2 A; accuracy was measured by complete relaxation matrix calculations, which yielded a sixth root R factor of 9.1 x 10(-)2 at 250 ms. The refined structure was a right-handed duplex, in which the benz[a]anthracene moiety intercalated from the major groove between C5.G18 and R,S,R,SA6.T17. In this orientation, the saturated ring of BA was oriented in the major groove of the duplex, with the aromatic rings inserted into the duplex such that the terminal ring of BA threaded the duplex and faced toward the minor groove direction. The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 8.8 A as compared to the value of 3.5 A normally observed for B-DNA between base pairs C5.G18 and R,S,R,SA6.T17. These two base pairs also buckled in opposite directions away from the intercalated BA moiety. The refined structure was similar to the (-)-(7S,8R,9S,10R)-N6-[10-(7,8,9, 10)-tetrahydrobenzo[a]pyrenyl)]-2'-deoxyadenosyl adduct of corresponding stereochemistry at X6 of the same oligodeoxynucleotide [Zegar, I. S., Kim, S. J., Johansen, T. N., Horton, P. J., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 6212-6224]. Both adducts intercalated toward the 5'-direction from the site of adduction. The similarities in solution structures were reflected in similar biological responses, when repair-deficient AB2480 Escherichia coli were transformed with M13mp7L2 DNA site-specifically modified with these two adducts.     

Interactions of aluminium(III) with glycerolphosphates and glycerophosphorylcholine

The complexation of aluminium(III) with glycerol-1-phosphate (G1P) and glycerol-2-phosphate (G2P) in aqueous solutions has been studied as a function of pH, by pH-potentiometry, 31P NMR spectroscopy and ESI mass spectrometry. Various mononuclear complexes (MLH(2)(3+), MLH(2+), ML(+), ML(2)H, ML(2)(-)) and polynuclear species (M(3)L(3)H(-1)(2+), M(3)L(2)H(-n)((n-5)-) with n=5, 6, 7, M(2)L(2)H(-1)(+) ) are formed in the system where the full protonated ligands are noted LH(2). NMR experiments clearly show that G1P and G2P already interact with Al(III) at pH 1. The potentiometric results are confirmed by ESI measurements and 31P NMR studies. No metal ion-induced deprotonation and coordination of the alcoholic-OH functions seem to occur during the complexation. The situation is very different for the glycerophosphorylcholine ligand (GPC identical with LH). Only the complex ML(3+) is formed in aqueous solution with a relatively low formation constant (K=5 at 37 degrees C). This species is clearly identified in 31P and 27Al NMR spectra. The complexation study as a function of the temperature allowed us to determine the thermodynamic parameters of the complex formation. The complexation is not governed by the reaction enthalpy that is found to be positive but by the entropy that is largely positive.     

NMR study of stacking interactions and conformational adjustments in the dinucleotide-carcinogen adduct 2'-deoxycytidylyl-(3----5)-2'-deoxy-8-(N-fluoren-2-ylac etamido)guanosi ne

The conformation and dynamics of the dinucleotide d-CpG modified at the C(8) position of the guanine ring by the carcinogen 2-(acetylamino)fluorene has been investigated by high-field 1H NMR spectroscopy. A two-state analysis of chemical shift data has enabled estimation of the extent of intramolecular stacking in aqueous solution as a function of temperature. The stacking, which is mostly fluorene-cytosine, is virtually complete in the low-temperature range. The 500-MHz 1H NMR spectrum consists of two subspectra near ambient temperatures due to a 14.3 +/- 0.3 kcal/mol barrier to internal rotation about the amide bond in the stacked form. A large barrier to internal rotation about the guanyl-nitrogen bond at C(8) has also been ascertained, but separate NMR subspectra were not detected due to the predominance of one of the torsional diastereomers (alpha' = 90 degrees) in the fully stacked state. Problems of self-association and chemical exchange were identified and overcome to enable analysis of the sugar-phosphate backbone conformation utilizing coupling constants. For the exocyclic C(4')-C(5') bond of the deoxyguanosine moiety, there is a high gauche+ (gamma = 60 degrees) conformer population, which is uncommon for a purine nucleotide with a syn orientation about the glycosyl bond. The gauche- conformation (gamma = 300 degrees), which is normally present in syn purine nucleotides in solution, was not detected. The exocyclic C(5')-O(5') torsion of the deoxy-guanosine moiety remains near the classical energy minimum (beta = 180 degrees) in the major stacked conformations. The sugar ring of the deoxycytidine moiety is predominantly in the C2'-endo conformation, while the deoxyguanosine ring is a mixture of conformations, one of which appears to be unusually puckered. The results support intercalation models of modified DNA and suggest a looped-out structure, with the modified guanine being the first base in the loop. Such structures could explain the relatively rapid rate of repair and the frame-shift mutations of this type of adduct.     

Metabolism of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate in the neuronal and glial compartments of the adult rat brain as detected by [(13)C, (2)H] NMR spectroscopy

Ex vivo ?(13)C, (2)H? NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose or (2-(13)C, 2-(2)H(3)) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution (13)C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2-(13)C, (2)H(3)) acetate or (1-(13)C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of (2)H-(13)C multiplets and (2)H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (4%), (2-(13)C) acetyl-CoA (3%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2-(13)C) acetyl-CoA (86%) and (2-(13)C, 2-(2)H) acetyl-CoA (5%). During the infusions of (2-(13)C, 2-(2)H(3)) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (36%), (2-(13)C, 2-(2)H) acetyl-CoA (27%) and (2-(13)C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2-(13)C) acetyl-CoA (11%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (16%). These results illustrate the potential of ?(13)C, (2)H? NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.     

Molecular recognition between oligopeptides and nucleic acids. Sequence specific binding of (4S)-(+)- and (4R)-(-)-dihydrokikumycin B to DNA deduced from 1H NMR, footprinting studies and thermodynamic data

The sequence specific binding of the antibiotic (4S)-(+)-dihydrokikumycin B and its (4R)-(-) enantiomer, [(S)-1 and (R)-1, respectively] to DNA were characterized by DNase I and MPE footprinting, calorimetry, UV spectroscopy, circular dichroism, and 1H NMR studies. Footprinting analyses showed that both enantiomers [(S)-1 and (R)-1] bind to AT-rich regions of DNA. 1H NMR studies (ligand induced chemical shift changes and NOE differences) of the dihydrkikumycins with d-[CGCAATTGCG]2 show unambiguously that the N to C termini of the ligands are bound to 5'-A5T6T7-3' reading from left to right. From quantitative 1D-NOE studies, the AH2(5)-ligand H7 distance of complex A [(S)-1 plus decamer (which is bound more strongly)] and complex B [(R)-1 and decamer] are estimated to be 3.8 +/- 0.3 A and 4.9 +/- 0.4 A, respectively. This difference in binding properties is reflected in the thermodynamic profiles of the two enantiomeric ligands determined by a combination of spectroscopic and calorimetric techniques. The binding free energies (delta G degrees) of (S)-1 and (R)-1 to poly d(AT).poly d(AT) at 25 degrees C are -31.8 and -29.3 kJ mol-1, respectively while the corresponding binding enthalpies (delta H degrees) are -11.3 and -0.8 kJ mol-1. These data permit the construction of models for the binding of the enantiomeric dihydrokikumycins to DNA and account for the more efficient binding of the natural (S) isomer to DNA.     

An NMR study of the lactonization of alpha-N-acetylneuraminyl-(2 --> 3)-lactose

The composition of the products formed by treatment of commercial alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc (3'-sialyllactose) with glacial acetic acid was investigated by 1H-13C one- and two-dimensional NMR spectroscopy and fast atom bombardment-mass spectrometry. The data confirmed that the major product of the reaction was alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc-(1c --> 2b)-lactone, which reverted to the starting material on standing in aqueous solution at ambient temperature, but for which complete NMR assignments are reported. The NMR data led to the tentative conclusion that the reaction also yielded small amounts of lactose, and alpha-Neu5Ac-(2 --> 3)-beta-D-Galp-(1 --> 4)-D-Glc-(1c --> 4b)-lactone which was stable in aqueous solution.     

Synthesis and in vivo evaluation of [O-methyl-11C] N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide as an imaging probe for 5-HT6 receptors

The serotonin receptor 6 (5-HT(6)) is implicated in the pathophysiology of cognitive diseases, schizophrenia, anxiety and obesity and in vivo studies of this receptor would be of value for studying the pathophysiology of these disorders. Therefore, N-[3,5-dichloro-2-(methoxy)phenyl]-4-(methoxy)-3-(1-piperazinyl)benzenesulfonamide (SB399885), a selective and high affinity (pK(i)=9.11) 5-HT(6) antagonist, has been radiolabeled with carbon-11 by O-methylation of the corresponding desmethyl analogue with [(11)C]MeOTf in order to determine the suitability of [(11)C]SB399885 to quantify 5-HT(6)R in living brain using PET. Desmethyl-SB399885 was prepared, starting from 1-(2-methoxyphenyl) piperazine hydrochloride, in excellent yield. The yield obtained for radiolabeling of [(11)C]SB399885 was 30±5% (EOS) and the total synthesis time was 30min at EOB. PET studies with [(11)C]SB399885 in baboon showed fast uptake followed by rapid clearance in the brain. Highest uptake of radioactivity of [(11)C]SB399885 in baboon brain were found in temporal cortex, parahippocampal gyrus, pareital cortex, amygdala, and hippocampus. Poor brain entry and inconsistent brain uptake of [(11)C]SB399885 compared to known 5-HT(6)R distribution limits its usefulness for the in vivo quantification of 5-HT(6)R with PET.     

2,2-Bis(ethoxycarbonyl)- and 2-(alkylaminocarbonyl)-2-cyano-substituted 3-(pivaloyloxy)propyl groups as biodegradable phosphate protections of oligonucleotides

Oligonucleotides bearing biodegradable phosphate protecting groups have been synthesized on a solid support. For this purpose, two dimeric building blocks, viz. 5'-O-(4,4'-dimethoxytrityl)-(R(P),S(P))-O(P)-[2,2-bis(ethoxycarbonyl)-3-(pivaloyloxy)propyl]-P-thiothymidylyl-(3',5')-thymidine 3'-[O-(2-cyanoethyl)-N,N-diisopropylphosphoramidite] (1) and 5'-O-(4,4'-dimethoxytrityl)-(R(P),S(P))-O(P)-[2-cyano-2-(2-phenylethylaminocarbonyl)-3-(pivaloyloxy)propyl]thymidylyl-(3',5')-thymidine 3'-(H-phosphonate) (2), were prepared. Phosphoramidite 1 was incorporated into an phosphorothioate oligothymidylate sequence on a base-labile hydroquinone-O,O'-diacetic acid linker (Q-linker) and on a photolabile 4-alkoxy-5-methoxy-2-nitrobenzyl carbonate linker (11). H-Phosphonate 2 was, in turn, incorporated into an oligothymidylate sequence only on the photolabile linker. Kinetics of the removal of the protecting groups by porcine liver esterase and subsequent retro aldol condensation/phosphate elimination were then studied. While the pro-oligonucleotide that contained only one phosphate protection gave the deprotected phosphorothioate oligonucleotide in a quantitative yield, the enzymatic step was markedly decelerated upon increasing the number of protection groups, and hence chain cleavage started to compete.     

Two-step regio- and stereoselective syntheses of [19F]- and [18F]-2-deoxy-2-(R)-fluoro-beta-D-allose

Replacement of specific hydroxyl groups by fluorine in carbohydrates is an ongoing challenge from chemical, biological, and pharmaceutical points of view. A rapid and efficient two-step, regio- and stereoselective synthesis of 2-deoxy-2-(R)-fluoro-beta-d-allose (2-(R)-fluoro-2-deoxy-beta-d-allose; 2-FDbetaA), a fluorinated analogue of the rare sugar, d-allose, is described. TAG (3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-d-arabino-hex-1-enitol or 3,4,6-tri-O-acetyl-d-glucal), was fluorinated in anhydrous HF with dilute F(2) in a Ne/He mixture or with CH(3)COOF at -60 degrees C. The fluorinated intermediate was hydrolyzed in 1N HCl and the hydrolysis product was purified by liquid chromatography and characterized by 1D (1)H, (13)C, and (19)F NMR spectroscopy as well as 2D NMR spectroscopy and mass spectrometry. In addition, (18)F-labeled 2-deoxy-2-(R)-fluoro-beta-d-allose (2-[(18)F]FDbetaA) was synthesized for the first time, with an overall decay-corrected radiochemical yield of 33+/-3% with respect to [(18)F]F(2), the highest radiochemical yield achieved to date for electrophilic fluorination of TAG. The rapid and high radiochemical yield synthesis of 2-[(18)F]FDbetaA has potential as a probe for the bioactivity of d-allose.     

Studies on tetrahydrofuranyl-5-fluorouracils. 2. NMR studies of 1-(tetrahydro-2-furanyl)-, 3-(tetrahydro-2-furanyl)-, and 1,3-bis(tetrahydro-2-furanyl)-5-fluorouracils.

Measurements of the 1H NMR spectra of the diastereoisomers of 1-(tetrahydro-2-furanyl)-5-fluorouracil, 3-(tetrahydro-2-furanyl)-5-fluorouracil, and 1,3-bis(tetrahydro-2-furanyl)-5-fluorouracil in the presence of tris[3-(2,2,2-trifluoro-1-hydroxyethylidene)-d-camphorato]europium(Eu(TFC)3) as a chiral shift reagent showed differences between the isomers in the chemical shift changes of the protons of C2'-H and C6-H etc.     

Synthesis and characterization of trans-[Pt(NH3)2Cl2] adducts of d(CCTCGAGTCTCC).d(GGAGACTCGAGG)

The reaction of trans-diamminedichloroplatinum(II) (trans-DDP), the inactive isomer of the anticancer drug cisplatin, with the single-stranded deoxydodecanucleotide d(CCTCGAGTCTCC) in aqueous solution at 37 degrees C was monitored by reversed-phase HPLC. Consumption of the dodecamer follows pseudo-first-order reaction kinetics with a rate constant of 1.25 (4) x 10(-4) s-1. Two intermediates, shown to be monofunctional adducts in which Pt is coordinated to the guanine N7 positions, were trapped with NH4(HCO3) and identified by enzymatic degradation analysis. These monofunctional adducts and a third, less abundant, one are rapidly removed from the DNA by thiourea under mild conditions. When allowed to react further, the monofunctional intermediates formed a single main product that was characterized by 1H NMR spectroscopy and enzymatic digestion as the bifunctional 1,3-intrastrand cross-link trans-[Pt(NH3)2[d(CCTCGAGTCTCC)-N7-G(5),N7-G(7]]). Binding of the trans-[Pt(NH3)2]2+ moiety to the guanosine N7 positions decreases the pKa at N1 and leads to destacking of the intervening A(6) base. The double-stranded trans-DDP-modified and unmodified DNAs were obtained by annealing the complementary strand to the corresponding single strands and then studied by 31P and 1H NMR and UV spectroscopy. trans-DDP binding does not induce large changes in the O-P-O bond or torsional angles of the phosphodiester linkages in the duplex, nor does it significantly alter the UV melting temperature. trans-DDP binding does, however, cause the imino protons of the platinated duplex to exchange rapidly with solvent by 50 degrees C, a phenomenon that occurs at 65 degrees C for the unmodified duplex. A structural model for the platinated double-stranded oligonucleotide was generated through molecular dynamics calculations. This model reveals that the trans-DDP bifunctional adduct can be accommodated within the double helix with minimal distortion of the O-P-O angles and only local disruption of base pairing and destacking of the platinated bases. The model also predicts hydrogen bond formation involving coordinated ammine ligands that bridge the two strands.     

Metabolism of 18:2(n - 6), 18:3(n - 3), 20:4(n - 6) and 20:5(n - 3) by the fungus Gaeumannomyces graminis: identification of metabolites formed by 8-hydroxylation and by w2 and w3 oxygenation.

The present study was aimed at developing a cell-free preparation of Gaeumannomyces graminis to biosynthesize w2-hydroxy, w3-hydroxy and related metabolites of essential fatty acids. 14C-labelled linoleic acid (18:2(n - 6)), linolenic acid (18:3(n - 3)), arachidonic acid (20:4(n - 6)) and eicosapentaenoic acid (20:5(n - 3)) were incubated with the cytosolic and microsomal fractions and NADPH. Significant metabolism was only found in the cytosol. The main products were purified by high-performance liquid chromatography and identified by gas chromatography-mass spectrometry (GC-MS). 18:2(n - 6) was metabolized mainly to 8-hydroxy-9,12-octadecadienoic acid (8-HODE), while the w2 and the w3 alcohols were formed in relatively small amounts. The absolute configuration of the 8-hydroxyl was found to be R by ozonolysis of the diastereoisomeric (-)-menthoxycarbonyl derivative of 8-HODE and GC-MS analysis. In analogy, 18:3(n - 3) was converted to 8-hydroxy-9,12,15-octadecatrienoic acid and to smaller amounts of the 15,16-diol (15,16-DiHODE). In contrast, 8-hydroxy metabolites of 20:4(n - 6) or 20:5(n - 3) could not be detected. 20:4(n - 6) was efficiently converted to 18(R)-hydroxyeicosatetraenoic acid (18(R)-HETE) and 19(R)-HETE and to traces of 17-HETE, while 20:5(n - 3) was mainly metabolized to the 17,18-diol (17,18-DiHETE) and to smaller amounts of the w2 alcohol. In conclusion, the cytosol of G. graminis can be used for stereoselective biosynthesis of some hydroxy metabolites of essential fatty acids.     

Synthesis, characterization and cytotoxic activity of substituted benzyl iminoether Pt(II) complexes of the type cis- and trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-R}2] (R=Me, OMe, F). X-ray structure of trans-[PtCl2{E-N(H)=C(OMe)CH2-C6H4-p-F}2

New substituted benzyl iminoether derivatives of the type cis- and trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-R}(2)] (R=Me (1a, 2a), OMe (3a, 4a), F (5a, 6a)) have been synthesized and characterized by elemental analyses, FT-IR spectroscopy and NMR techniques. The iminoether ligands are in the E configuration, which is stable in solution and in the solid state, as confirmed by the (1)H NMR data. Complex trans-[PtCl(2){E-N(H)C(OMe)CH(2)-C(6)H(4)-p-F}(2)] (6a) was also characterized by an X-ray diffraction study. Complexes 1a-6a have been tested against a panel of human tumor cell lines in order to evaluate their cytotoxic activity. cis-Isomers were significant more potent than the corresponding trans-isomers against all tumor cell lines tested; moreover, complexes 1a and 5a showed IC(50) values from about 2-fold to 6-fold lower than those exhibited by cisplatin, used as reference platinum anticancer drug.     

Facile transition between 3(10)- and alpha-helix: structures of 8-, 9-, and 10-residue peptides containing the -(Leu-Aib-Ala)2-Phe-Aib- fragment.

A structural transition from a 3(10)-helix to an alpha-helix has been characterized at high resolution for an octapeptide segment located in 3 different sequences. Three synthetic peptides, decapeptide (A) Boc-Aib-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, nonapeptide (B) Boc-Trp-(Leu-Aib-Ala)2-Phe-Aib-OMe, and octapeptide (C) Boc-(Leu-Aib-Ala)2-Phe-Aib-OMe, are completely helical in their respective crystals. At 0.9 A resolution, R factors for A, B, and C are 8.3%, 5.4%, and 7.3%, respectively. The octapeptide and nonapeptide form ideal 3(10)-helices with average torsional angles phi(N-C alpha) and psi(C alpha-C') of -57 degrees, -26 degrees C and -60 degrees, -27 degrees for B. The 10-residue peptide (A) begins as a 3(10)-helix and abruptly changes to an alpha-helix at carbonyl O(3), which is the acceptor for both a 4-->1 hydrogen bond with N(6)H and a 5-->1 hydrogen with N(7)H, even though the last 8 residues have the same sequence in all 3 peptides. The average phi, psi angles in the decapeptide are -58 degrees, -28 degrees for residues 1-3 and -63 degrees, -41 degrees for residues 4-10. The packing of helices in the crystals does not provide any obvious reason for the transition in helix type. Fourier transform infrared studies in the solid state also provide evidence for a 3(10)- to alpha-helix transition with the amide I band appearing at 1,656-1,657 cm-1 in the 9- and 10-residue peptides, whereas in shorter sequences the band is observed at 1,667 cm-1.     

Crystal and molecular structure of (E)-5-(2-bromovinyl-2''-deoxyuridine)

(E)-5-(2-bromovinyl-2'-deoxyuridine) crystallizes in the space group P2(1) with a = 12.976(1), b = 4.800(1), c = 20.385(2) A, beta = 96.88(1) degrees, Z = (two molecules a and b in the asymmetric unit). The structure has been determined by the use of 2400 diffractometer reflexions and refined by least-squares to R of 0.053. Conformational features of both molecules a and b resemble those of thymidine. The ribofuranose rings assume the rare C(3')-exo form observed also in thymidine. Similarly, the torsion angles around the glycosidic bonds (mean = 40(1) and 56(1) degrees fall in the anti range. In each molecule the best plane of the 2-bromovinyl moiety is bent out of the least-squares plane of the pyrimidine base by 6 degrees, so that the positively charged C(8)-H(8) group can donate an intramolecular hydrogen bond to 0(4) atom. Eight strong and weak intermolecular hydrogen bridges are built up between the symmetry independent and related molecules forming a complicated three dimensional hydrogen bond network.