Confocal fluorescence imaging of photosensitized DNA denaturation in cell nuclei

The double-stranded helical structure of DNA is maintained in part by hydrogen bonds between strands and by stacking interactions between adjacent purine and pyrimidine bases in one strand. The transition (denaturation) from a double-stranded (ds) to a single-stranded (ss) form can be induced in isolated DNA or fixed cells by exposure to elevated temperatures, alkali or acids, aprotic or nonpolar solvents or some drugs. We report here that DNA denaturation can occur in situ in cell nuclei as a result of interaction between light and an intercalated dye, acridine orange or ethidium bromide. This DNA photodenaturation was probed using metachromatic properties of acridine orange and imaged by fluorescence confocal microscopy. Furthermore, an empirical kinetic model was developed to separate changes of acridine orange luminescence intensities caused by photobleaching from those that were a result of DNA denaturation. We investigated the influence of oxygen on these phenomena and propose a mechanism by which photodenaturation may occur.     

Faster dissociation: measured rates and computed effects on barriers in aryl halide radical anions

Carbon-halogen bond dissociation rates for a series of aryl halide radical anions (ArX-: X = Cl, Br) in NMP were measured at room temperature by pulse radiolysis with 10-11 s time resolution. To obtain accurate dissociation rates, care was taken to measure and correct for competing decay channels. The observed rates correlated well with activation energies computed in the gas phase by density functional (DFT) calculations. The rates did not correlate well with electron affinities or dissociation energies obtained by the same computational methods, although such correlations are reported in the literature and are expected on the basis of simple models. The calculations also found that the transition state structures had bent carbon-halogen bonds. Bending enables large reductions of the activation energies by an electronic effect involving mixing of phi* and sigma* states. This bending-induced mixing is computed to increase the dissociation rates by a few orders of magnitude and is thus essential to understanding these reactions.     

Identification of ligands for RNA targets via structure-based virtual screening: HIV-1 TAR

Binding of the Tat protein to TAR RNA is necessary for viral replication of HIV-1. We screened the Available Chemicals Directory (ACD) to identify ligands to bind to a TAR RNA structure using a four-step docking procedure: rigid docking first, followed by three steps of flexible docking using a pseudobrownian Monte Carlo minimization in torsion angle space with progressively more detailed conformational sampling on a progressively smaller list of top-ranking compounds. To validate the procedure, we successfully docked ligands for five RNA complexes of known structure. For ranking ligands according to binding avidity, an empirical binding free energy function was developed which accounts, in particular, for solvation, isomerization free energy, and changes in conformational entropy. System-specific parameters for the function were derived on a training set of RNA/ligand complexes with known structure and affinity. To validate the free energy function, we screened the entire ACD for ligands for an RNA aptamer which binds l-arginine tightly. The native ligand ranked 17 out of ca. 153,000 compounds screened, i.e., the procedure is able to filter out >99.98% of the database and still retain the native ligand. Screening of the ACD for TAR ligands yielded a high rank for all known TAR ligands contained in the ACD and suggested several other potential TAR ligands. Eight of the highest ranking compounds not previously known to be ligands were assayed for inhibition of the Tat-TAR interaction, and two exhibited a CD₅₀ of ca. 1 M.     

The enantiospecific,stereospecific total synthesis of the ring-A oxygenated sarpagine indole alkaloids (+)-majvinine, (+)-10-methoxyaffinisine,and (+)-N(a)-methylsarpagine,as well as the total synthesis of the alstonia bisindole alkaloid macralstonidine

The first stereospecific, enantiospecific total synthesis of the ring-A oxygenated sarpagine indole alkaloids (+)-N(a)-methylsarpagine (8), (+)-majvinine (14), and (+)-10-methoxyaffinisine (49), as well as the first total synthesis of the Alstonia bisindole alkaloid macralstonidine (9), has been accomplished. This approach employed the Sch?llkopf chiral auxiliary for the stereospecific construction of the desired d-(+)-tryptophan unit required for the asymmetric Pictet-Spengler reaction. In addition, the strategy was doubly convergent for the enolate-mediated Pd(0) coupling process and the asymmetric Pictet-Spengler reaction can be employed to synthesize both macroline (2) and N(a)-methylsarpagine (8), the coupling of which provides macralstonidine (9). This approach to ring-A substituted alkoxyindole alkaloids should find wide application for the synthesis of other alkaloids for it is stereospecific and either enantiomer can be prepared with ease.     

Synthesis and resolution of (R,R)-(+/-)-1,1,4,7,10,10-hexaphenyl-1,10-diarsa-4,7-diphosphadecane: new ligand for the stereoselective self-assembly of dicopper(I), disilver(I), and digold(I) helicates

Diphenylvinylarsine oxide reacts with 1,2-bis(phenylphosphino)ethane in the presence of potassium tert-butoxide to give the anti-Markovnikov product (R,R)-(+/-)/(R,S)-1,1,4,7,10,10-hexaphenyl-1,10-diarsa-4,7-diphosphadecane dioxide-1AsO,10AsO, which, upon reduction with HSiCl(3)/NEt(3) in boiling acetonitrile, affords in 84% overall yield the di(tertiary arsine)-di(tertiary phosphine) (R,R)-(+/-)/(R,S)-diphars. After separation of the diastereomers by fractional crystallization, the (R,R)-(+/-) form of the ligand was resolved by metal complexation with (+)-di(mu-chloro)bis[(R)-1-[1-(dimethylamino)ethyl]-2-phenyl-C(2),N]dipalladium(II): (R,R)-diphars, mp 87-88 degrees C, has [alpha](D)(21) = -18.6 (c 1.0, CH(2)Cl(2)); (S,S)-diphars has [alpha](D)(21) = +18.4 (c 1.0, CH(2)Cl(2)). The crystal and molecular structures of the complexes (M)-[M(2)[(R,R)-diphars](2)](PF(6))(2) (M = Cu, Ag, Au) have been determined: [M-(S(Cu),S(Cu))]-(-)-[Cu(2)[(R,R)-diphars](2)](PF(6))(2), orthorhombic, P2(1)2(1)2(1) (No. 19), a = 16.084(3) A, b = 18.376(3) A, c = 29.149(6) A, Z = 4; [M-(S(Ag),S(Ag))]-(+)-[Ag(2)[(R,R)-diphars](2)](PF(6))(2), triclinic, P1, a = 12.487(2) A, b = 12.695(4) A, c = 27.243(4) A, alpha = 92.06 degrees, beta = 95.19 degrees, gamma = 98.23 degrees, Z = 2; [M-(S(Au),S(Au))]-(-)-[Au(2)[(R,R)-diphars](2)](PF(6))(2), orthorhombic, P2(1)2(1)2(1) (No. 19), a = 16.199(4) A, b = 18.373(4) A, c = 29.347(2) A, Z = 4. In the copper(I) and gold(I) helicates, each ligand strand completes 1.5 turns of an M helix in a parallel arrangement about the two chiral MAs(2)P(2) stereocenters of S configuration. The unit cell of the silver(I) complex contains one molecule each of the parallel helicate of M configuration and the conformationally related double alpha-helix of M configuration in which each ligand strand completes 0.5 turns of an M helix about two metal stereocenters of S configuration. Energy minimization calculations of the three structures with use of the program SPARTAN 5.0 gave results that were in close agreement with the core structures observed.     

Synthesis and conformational properties of 2′-deoxy-2′-methylthio-pyrimidine and -purine nucleosides: Potential antisense applications

A convenient and shorter synthesis of 2′-deoxy-2′-methylthiouridine analogs 5 , ?5-methyluridine 6 , -cyti-dine 15 , ?5-methylcytidine 16 , -adenosine 27 and -guanosine 34 was accomplished. Successful conversion of ribonucleosides (5-methyl U, U, A, G) into the corresponding 2′-substituted nucleosides involves nucleophilic displacement (S_N2) of an appropriate leaving group at the 2′-position by methanethiol, a soft nucleophile. Reaction between 2,2′-anhydrouridine and methanethiol in the presence of N¹,N¹,N³,N³-tetramethylguani-dine in N,N-dimethylformamide gave 5 , in 75% yield. Preparation of 6 by a similar route was described. Acylated 5 and 6 were transformed into their triazole derivatives, which on ammonolysis furnished 15 and 16 , respectively in good yield. Similarly, tetraisopropyldisiloxanyl (TIPS) protected 2′-O-aratriflates- of-adenosine and -guanosine reacted with methanethiol in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene at - 25°, followed by deblocking of the TIPS protecting group furnished 27 and 34 , respectively. The confor-mational flexibility (N/S equilibrium) of the sugar moiety in nucleosides 5 , 15 , 27 and 34 was studied utilizing nmr spectroscopy, suggesting that the 2′-methylthio group influenced the sugar conformation to adopt a rigid S-pucker in all cases. The extra stiffness of the sugar moiety in these analogs is believed to be due to the electronegativity of the substituent and the steric bulk. The usefulness of these nucleosides to prepare uniformly modified 2′-deoxy-2′-methylthio oligonucleotides for antisense therapeutics is proposed.     

Applications of fourier transforms in molecular orbital theory. Calculation of optical properties and tables of two-center one-electron integrals

Fourier transform methods initiated by Geller and Harris are applied to the calculation of optical properties of molecules. Tables of one-electron two-center integrals needed for the accurate computation of molecular absorption and optical activity are calculated by the Fourier transform method. A general theorem is derived which allows the angular part of the integrals to be treated by means of projection operators. The radial parts of the integrals are treated by the methods of Harris. The results are obtained in a simple closed form which avoids the usual transformation to local coordinates. The two-center integrals evaluated include matrix elements of the momentum operator, the dipole moment operator, the tensor operator , the quadrupole moment operator, and the angular momentum operator. These are evaluated between 1s, 2s, and 2p Slater-type atomic orbitals located on different atoms. The results are expressed as functions of the Slater exponents and of the relative coordinates of the two atoms.     

A mechanistic study of electrospray mass spectrometry: Charge gradients within electrospray droplets and their influence on ion response

A model has been developed to account qualitatively for the effects of ion pairing, surface activity, and electrophoretic mobility in electrospray mass spectrometry. The model is tested with various salt and amino acid mixtures. The data suggest that the axial charge gradient arising from electrophoretic separation at droplet genesis may persist within the electrosprayed droplets at least until the first droplet fission, accounting for the field dependence of detected ion clustering of quaternary ammonium salts and for the relatively field-invariant charge distribution of horse heart myoglobin samples.     

A synthesis of 2-β-D-ribofuranosyl-4-selenazolecarboxamide (selenazofurin) and certain N-substituted amide derivatives suitable for large scale syntheses

A new process suitable for large scale synthesis of the antitumor-antiviral agent, 2-β-D-ribofuranosyl-4-selenazolecarboxamide (selenazofurin, 1 ), has been developed. Thus, 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose ( 3 ) was converted with cyanotrimethylsilane and stannic chloride to the crystalline 2,5-anhydro-3,4,6-tri-O-benzoyl-β-D-allononitrile ( 4 ) without chromatography. Cyanosugar 4 in ethanol was treated with hydrogen selenide gas to afford stereospecifically the unstable 2,5-anhydro-3,4,6-tri-O-benzoyl-β-D-allonoselenoamide ( 5 ) which was converted in situ by ethyl bromopyruvate to the stable ethyl 2-(2,3,5-tri-O-benzoyl-β-D-ribofuranosyl)-4-selenazolecarboxylate ( 6). Selenazole ethyl ester 6 was deprotected with sodium methoxide affording methyl 2-β-D-ribofuranosyl-4-selenazolecarboxylate ( 7 ) which was aminated with ammonia to provide selenazofurin ( 1 ) or with other amines to provide N-substituted selenazofurin amides.     

Total synthesis of indole and dihydroindole alkaloids. VIII. Studies on the synthesis of bisindole alkaloids in the vinblastine-vincristine series. The chloroindolenine approach.

Studies on the syntheses of 18′-epi-4′-deoxo-4′-epivinblastine (IX, R = CO₂CH₃; R₁ = H), 18′-decarbomethoxy-18′-epi-4′-deoxo-4′-epivinblastine (IX, R = R₁ = H) and related analogues are described. The synthetic method employs a coupling reaction involving chloroindolenine derivatives of the cleavamine series (for example, III) with vindoline (V) under acidic conditions. The complete structures, including absolute configuration, of the resulting dimers are established by a combination of chemical and spectroscopic techniques, including X-ray analysis.