Shapes of benz[a]anthracenes: the crystal and molecular structure of 2-methylbenz[a]anthracene

The crystal structure of 2-methylbenz[a]anthracene (2-MBA), the least carcinogenically active of the monomethylbenz[a]anthracenes, has been determined by application of direct methods to single-crystal X-ray diffractometric data and refined by least squares to R = 0.033 (Rw = 0.035). Deviations of the carbon atoms from the mean molecular plane are much smaller than in the rather more active 1-MBA; in 2-MBA, the benzo-ring A is inclined at about 2 degrees to each of the three rings in the anthracene moiety and even the methyl carbon atom is displaced by only 0.07 A from the ring-carbon atom plane of 2-MBA (and by 0.01 A from the ring-A plane). As in other MBA, the shortest C-C bond in this accurately determined structure is at the K-region (C(5)-C(6) = 1.330(3) A) but three other bonds are short; C(8)-C(9) = 1.347(4), C(10)-C(11) = 1.353(3) and the M-region bond C(3)-C(4) = 1.359(4) A (0.003 A longer if corrected for rigid-body librations). The 2-methyl group appears to take up two orientations with one trio of hydrogen positions more favored than the other.     

Shapes of benz[a]anthracenes: the crystal and molecular structure of 6-methylbenz[a]anthracene

The crystal structure of 6-methylbenz[a]anthracene (6-MBA), a more potent carcinogen than the other K-region monomethyl-substituted benz[a]anthracene (5-MBA), has been determined by application of direct methods to single-crystal X-ray diffractometric data and refined by least squares to R = 0.047 (Rw = 0.053). Deviations of the carbon atoms from planarity are very small with even the methyl carbon displaced by only 0.05 A from the mean molecular plane. The benzo-ring A is inclined at only about 1 1/2 degrees to each of the three rings in the anthracene moiety, i.e. 6-MBA is one of the most nearly planar benz[a]anthracenes. The K-region bond C(5)-C(6) = 1.328(6) A and two other short bonds are C(8)-C(9) = 1.341(7) and C(10)-C(11) = 1.361(7) A in the anthracene D ring.     

A new crystal form of beta-cyclodextrin-ethanol inclusion complex: channel-type structure without long guest molecules

A new crystal form of beta-cyclodextrin (beta-CD)[bond]ethanol[bond]dodecahydrate inclusion complex [(C(6)H(10)O(5))(7).0.3C(2)H(5)OH.12H(2)O] belongs to monoclinic space group C2 (form II) with unit cell constants a=19.292(1), b=24.691(1), c=15.884(1) A, beta=109.35(1) degrees. The beta-CD macrocycle is more circular than that of the complex in space group P2(1) [form I: J. Am. Chem. Soc. 113 (1991) 5676]. In form II, a disordered ethanol molecule (occupancy 0.3) is placed in the upper part of beta-CD cavity (above the O-4 plane) and is sustained by hydrogen bonding to water site W-2. In form I, an ethanol molecule located below the O-4-plane is well ordered because it hydrogen bonds to surrounding O-3[bond]H, O-6[bond]H groups of the symmetry-related beta-CD molecules. In the crystal lattice of form I, beta-CD macrocycles are stacked in a typical herringbone cage structure. By contrast, the packing structure of form II is a head-to-head channel that is stabilized at both O-2/O-3 and O-6 sides of each beta-CD by direct O(CD)...O(CD) and indirect O(CD)...O(W)...(O(W))...O(CD) hydrogen bonds. The 12 water molecules are disordered in 18 positions both inside the channel-like cavity of beta-CD dimer (W-1[bond]W-6) and in the interstices between the beta-CD macrocycles (W-7[bond]W-18). The latter forms a cluster that is hydrogen bonded together and to the neighboring beta-CD O[bond]H groups.     

Thiolate exchange in [TmR]ZnSR' complexes and relevance to the mechanisms of thiolate alkylation reactions involving zinc enzymes and proteins

The zinc and cadmium thiolate complexes [TmBut]MSCH2C(O)N(H)Ph (M = Zn, Cd) may be obtained via treatment of the respective methyl complex [TmBut]MMe with PhN(H)C(O)CH2SH. The molecular structure of [TmBut]ZnSCH2C(O)N(H)Ph has been determined by X-ray diffraction, thereby demonstrating the presence of an intramolecular N-H S hydrogen bond between the amide N-H group and thiolate sulfur atom. [TmBut]ZnSCH2C(O)N(H)Ph mimics the function of the Ada DNA repair protein by undergoing alkylation with MeI to give [TmBut]ZnI and MeSCH2C(O)N(H)Ph. A series of crossover experiments and 1H NMR magnetization transfer studies establish that thiolate exchange between [TmR]ZnSR' derivatives is facile in this system, an observation that supports the previous suggestion that the alkylation of [TmPh]ZnSCH2C(O)N(H)Ph by MeI may proceed via a sequence that involves dissociation of [PhN(H)C(O)CH2S]-.     

Shapes of carcinogenic benz[alpha]anthracenes: an x-ray crystal structure analysis of 12-methylbenz[alpha]anthracene.

The crystal structure of the moderately active carcinogen 12-methylbenz[alpha]anthracene (12-MBA) has been determined by application of direct methods to X-ray single-crystal diffraction data. Least-squares refinement to a residual R = 0.09 over 929 independent reflections enabled carbon positions to be established with apparent e.s.d.s. of atomic coordinates about 0.008 A. Deviation from planarity is exemplified by the 15.5 degrees inclination of the benz ring (A) to the anthracene nucleus and by the 0.89 A distance of the methyl carbon out of the best plane through the whole benzanthracene nucleus. Comparison with the structure of the highly carcinogenic 7,12-dimethylbenz[alpha]anthracene (7,12-DMBA), and with the recently solved structures of the weak carcinogen 1-MBA and the extremely weak carcinogen 1,12-DMBA, shows a close similarity in the anthracene parts; in 1-MBA, and 1,12-DMBA, the phenanthrenic K-region bond is close to 1.34 A and the M-region bond about 1.38 A. In 12-MBA, overcrowding in the 'bay' region causes the central anthracene ring C and the benz ring A each to be bent about 10 degrees in opposite directions from the phenanthrenic B ring, much as in 1-MBA and 7,12-DMBA, but less than in 1,12-DMBA; the 12-methyl carbon lies about the same distance (0.55 A) above the anthracene plane in 12-MBA as in 1,12-MBA and 7,12-DMBA.     

Synthesis, structural, physico-chemical and biological properties of new palladium(II) complexes with 2,6-dimethyl-4-nitropyridine

This paper describes the synthesis and properties of two new palladium(II) complexes with 2,6-dimethyl-4-nitro-pyridine (dmnp): mononuclear [Pd(dmnp)2Cl2] and dinuclear [Pd2(dmnp)2Cl4]. Complexes were characterized on the basis of chemical and chromatographic analyses, MS and conductometric measurements, as well as by IR and NMR (1H and 13C) spectral studies. The crystal structures of ligand and mononuclear complex, trans-dichlorobis(2,6-dimethyl-4-nitro-pyridine)palladium(II), were determined by three-dimensional X-ray methods. The crystals of both compounds are monoclinic, space groups P21/c with a=19.075(4), b=5.419(1), c=15.045(3) A and beta=108.15(3)degrees for (dmnp), and a=7.544(2), b=14.509(3), c=8.032(2) A and beta=90.32(3)degrees for [Pd(dmnp)2Cl2]. In the (dmnp) there are two crystallographically independent molecules in the unit cell. The nitro groups and methyl C atoms are coplanar with the ring plane. The hydrogen bond of the type C-H...O links the molecules into pairs around center of symmetry. These dimers are held together by contacts of the van der Waals type. In the crystal structure of [Pd(dmnp)2Cl2] the Pd atom lies on an inversion center and is four-coordinated by two pyridine N atoms and by two Cl atoms in trans positions. The coordination geometry is square-planar, with Pd-N and Pd-Cl distances of 2.033(2) and 2.311(1) A, respectively. The two pyridine rings are mutually parallel, but they are twisted from the PdN2Cl2 coordination plane by about 88.5degrees. The preliminary assessments of anti-tumor properties of both complexes and ligand were evaluated as in vitro anti-proliferative activity in four human cancer cell lines: SW707 (adenocarcinoma of the rectum), T47D (breast cancer), HCV (bladder cancer) and A549 (non-small cell lung carcinoma). The [Pd(dmnp)2Cl2] exhibits strong cytotoxic activity against all cell lines whereas the free ligand and dinuclear [Pd2(dmnp)2Cl4] are only moderate active.     

Hydrogen bonding in the crystal structure of bis[3-([thiomethyl]methyl)pyrazole]copper(II) perchlorate

The ability of a metal-coordinated pyrazole to engage in hydrogen bonding has been explored by synthesis of the title complex, bis[3-([thiomethyl]methyl)pyrazole]copper(II) perchlorate (3). The coordination in 3 can be described as pseudo-octahedral, with two relatively tightly-bound 3-[(thiomethyl)methyl]pyrazole ligands occupying the equatorial plane, forming a [CuN(2)S(2)](2+) unit with the S donors mutually trans to each other. The axial positions are each filled by a weakly bound perchlorate counterion, one oxygen of which forms a hydrogen bond with the pyrazole N-H moiety on an adjacent [CuN(2)S(2)](2+) unit.     

Crystal and molecular structure of methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside

The cellulose model compound methyl 4-O-methyl-beta-D-glucopyranosyl-(1-->4)-beta-D-glucopyranoside (6) was synthesised in high overall yield from methyl beta-D-cellobioside. The compound was crystallised from methanol to give colourless prisms, and the crystal structure was determined. The monoclinic space group is P2(1) with Z=2 and unit cell parameters a=6.6060 (13), b=14.074 (3), c=9.3180 (19) A, beta=108.95(3) degrees. The structure was solved by direct methods and refined to R=0.0286 for 2528 reflections. Both glucopyranoses occur in the 4C(1) chair conformation with endocyclic bond angles in the range of standard values. The relative orientation of both units described by the interglycosidic torsional angles [phi (O-5' [bond] C-1' [bond] O-4 [bond] C-4) -89.1 degrees, Phi (C-1' [bond] O-4 [bond] C-4 [bond] C-5) -152.0 degrees] is responsible for the very flat shape of the molecule and is similar to those found in other cellodextrins. Different rotamers at the exocyclic hydroxymethyl group for both units are present. The hydroxymethyl group of the terminal glucose moiety displays a gauche-trans orientation, whereas the side chain of the reducing unit occurs in a gauche-gauche conformation. The solid state (13)C NMR spectrum of compound 6 exhibits all 14 carbon resonances. By using different cross polarisation times, the resonances of the two methyl groups and C-6 carbons can easily be distinguished. Distinct differences of the C-1 and C-4 chemical shifts in the solid and liquid states are found.     

Ring conformation of cyclic octapeptide cyclo (L-Pro-Sar)4 in the crystalline state

A single-crystal X-ray diffraction analysis has been made of the structure of the cyclic octapeptide cyclo(L-Pro-Sar)4. The material [C32H48O8N8 X (21/4) H2O X (1/2) CH3OH, Mr = 799.43] crystallizes in the monoclinic space group C2 with cell dimensions a = 14.544 (3), b = 11.902 (2), c = 14.064 (3), and beta = 122.26 (2) degrees (lambda = 1.54178 A, T = 293 K). The final R value for the 1980 observed reflections is 0.079. The ring conformation has the peptide bond sequences of cis-cis-trans-trans-cis-cis-trans-trans (Pro-Sar-Pro peptide bond linkages are cis-cis- or trans-trans). The pyrrolidine rings in the four proline residues take an envelope form in which the gamma-carbon atom deviates from the plane of the remaining four atoms in the ring.     

k-Binding and Degradation of [3H]Dynorphin A (1–8) and [3H]Dynorphin A (1–9) in Suspensions of Guinea Pig Brain Membranes

Following incubation of [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9) with suspensions of guinea pig brain membranes, analysis of the supernatants by HPLC has shown that both peptides are degraded at 25 degrees C and at 0 degrees C. Bestatin and captopril reduce degradation at 0 degrees C but for a similar degree of protection at 25 degrees C arginine-containing dipeptides are also required. The effects of these peptidase inhibitors on the degradation profiles indicate that [3H]dynorphin A (1-8) has three main sites of cleavage: the Tyr1-Gly2, Arg6-Arg7, and Leu5-Arg6 bonds. With [3H]dynorphin A (1-9) as substrate the Arg7-Ile8 and Ile8-Arg9 bonds are also liable to cleavage. In binding assays, in contrast to the effects of peptidase inhibitors on the degradation of unbound [3H]dynorphin A (1-8) and [3H]dynorphin A (1-9), bestatin and captopril have little effect on the binding characteristics of the tritiated dynorphin A fragments at the kappa-site at 0 degrees C. However, at 25 degrees C binding is low in the absence of peptidase inhibitors. When binding at mu- and delta-sites is prevented, the maximal binding capacities of [3H]dynorphin A (1-8), [3H]dynorphin A (1-9), and [3H](-)-bremazocine at the kappa-site are similar; [3H]dynorphin A (1-9) has 5-10 times higher affinity for the kappa-site than [3H]dynorphin A (1-8). Comparison of the effects of peptidase inhibitors on unbound dynorphin A fragments with their effects in binding assays suggests that the bound peptides are protected from the action of peptidases.     

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.     

X-ray crystallographic conformational study of 5'-O-[N-(L-alanyl)-sulfamoyl]adenosine, a substrate analogue for alanyl-tRNA synthetase

In order to elucidate the substrate specificity of alanyl-tRNA synthetase, 5'-O-[N-(L-alanyl)sulfamoyl]adenosine (Ala-SA), an analogue of alanyl-AMP, was chemically synthesized. Its binding ability is similar to that of the substrate based on the inhibitory activity for the aminoacylation of alanyl-tRNA synthetase. Taking advantage of the stable sulfamoyl bond of Ala-Sa, compared with the highly labile aminoacyl bond of alanyl-AMP, the molecular conformation of the former inhibitor was studied by X-ray single crystal analysis. Crystal data are as follows: C13H19N7O7S.2H2O, space group C2, a = 39.620(6), b = 5.757(1), c = 20.040(3) A, beta = 117.2(1) degrees, V = 4065(9) A3, Z = 8, and final R = 0.065 for 2785 independent reflections of F(2)0 greater than or equal to 2 sigma (F0)2. In the crystal, the molecule is in a zwitterionic state with the terminal amino group protonated and sulfamoyl group deprotonated, and takes an open conformation, where the L-alanine moiety is located far from the adenosine moiety with gauche/trans and trans orientations about the exocyclic C(4')-C(5') and C(5')-O(5') bonds, respectively. The conformation of the adenosine moiety is anti for the glycosyl bond and C(3')-endo for the ribose puckering, and alanine is in the usually observed trans region for the psi torsion angle. The molecular dimensions of the sulfamoyl group are nearly the same as those of the phosphate group. The biological significance of the observed Ala-SA conformation is discussed in relation with the molecular conformation of tyrosyl-AMP complexed with tyrosyl-tRNA synthetase.     

Rates of oxygen and hydrogen exchange as indicators of TPQ cofactor orientation in amine oxidases.

This study presents the first detailed examination by resonance Raman (RR) spectroscopy of the rates of solvent exchange for the C5 and C3 positions of the TPQ cofactor in several wild-type copper-containing amine oxidases and mutants of the amine oxidase from Hansenula polymorpha (HPAO). On the basis of crystal structure analysis and differing rates of C5 [double bond] O and C3 [bond] H exchange within the enzyme systems, but equally rapid rates of C5 [double bond] O and C3 [bond] H exchange in a TPQ model compound, it is proposed that these data can be used to determine the TPQ cofactor orientation within the active site of the resting enzyme. A rapid rate of C5 [double bond] O exchange (t(1/2) < 30 min) and a slow (t(1/2) = 6 h) to nonexistent rate of C3 [bond] H exchange was observed for wild-type HPAO, the amine oxidase from Arthrobacter globiformis, pea seedling amine oxidase at pH 7.1, and the E406Q mutant of HPAO. This pattern is ascribed to a productive TPQ orientation, with the C5 [double bond] O near the substrate-binding site and the C3 [bond] H near the Cu. In contrast, a slow rate of C5 [double bond] O exchange (t(1/2) = 1.6-3.3 h) coupled with a fast rate of C3 [bond] H exchange (t(1/2) < 30 min) was observed for the D319E and D319N catalytic base mutants of HPAO and for PSAO at pH 4.6 (t(1/2) = 4.5 h for C5 [double bond] O exchange). This pattern identifies a flipped orientation, involving 180 degrees rotation about the C alpha-C beta bond, which locates the C3 [bond] H near the substrate-binding site and the C5 double bond] O near the Cu. Finally, fast rates of both C5 [double bond] O and C3 [bond] H exchange (t(1/2) < 30 min) were observed for the amine oxidase from Escherichia coli and the N404A mutant of HPAO, suggesting a mobile cofactor, with multiple TPQ orientations between productive and flipped. These results demonstrate that opposing sides of the TPQ ring possess different degrees of solvent accessibility and that the rates of C5 [double bond] O and C3 [bond] H exchange can be used to predict the TPQ cofactor orientation in the resting forms of these enzymes.     

Characterization df a [3H]Glycine Recognition Site as a Modulatory Site of the N-Methyl-D-Aspartate Receptor Complex

A [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM) has been identified, having characteristics expected of a modulatory component of the N-methyl-D-aspartate receptor complex. Incubation of SPM with [3H]glycine for 10 min at 2 degrees C results in saturable, reversible binding with a KD of 0.234 microM and a Bmax of 9.18 pmol/mg. A pharmacological analysis of this binding site indicates that D-serine (Ki = 0.27 microM), D-alanine (Ki = 1.02 microM), and D-cycloserine (Ki = 2.33 microM) are potent inhibitors of binding, whereas the corresponding L isomers have significantly less activity (Ki = 25.4 microM, 15.9 microM, and greater than 100 microM, respectively). Inactive at concentrations of up to 100 microM were strychnine, L-valine, N,N-dimethylglycine, aminomethylphosphonate, and aminomethylsulfonate. The active compounds were analyzed further for their ability to stimulate [3H]1-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) binding to Triton X-100-washed SPM. Results indicate that the affinity of the compounds for the [3H]glycine recognition site correlates with the ability of these analogues to stimulate [3H]TCP binding.     

Effects of thioamide substitution for the enkephalin conformation. Crystal structure of Boc-Tyr-Gly-Gly-Phe psi [CSNH]Leu-OBzl

The crystals of Boc-Tyr-Gly-Gly-Phe psi[CSNH]Leu-OBzl monohydrate (C40H51N5O8S.H2O), a monothionated Leu-enkephalin analogue, were obtained with space group P2(1), a = 12.616(3), b = 9.347(2), c = 18.548(5) A, beta = 96.31(4) degrees. The structure was elucidated by X-ray diffraction analysis, and refined to the R value of 0.091 for the observed 3294 reflections. Two antiparallel molecules related by a pseudo twofold symmetry were stabilized to each other by four intermolecular hydrogen bonds. The molecular conformation was bent at the Phe residue, and the extended moiety of the Tyr-Gly-Gly fragment was almost perpendicular to that of the Phe-Leu residues. Consequently the molecule, as a whole, formed an L-shape conformation with a slightly left-handed helicity.     

Exceptional stability of a [2Fe-2S] ferredoxin from hyperthermophilic bacterium Aquifex aeolicus

Aquifex aeolicus is the only hyperthermophile that is known to contain a plant- and mammalian-type [2Fe-2S] ferredoxin (Aae Fd1). This unique protein contains two cysteines, in addition to the four that act as ligands of the [2Fe-2S] cluster, which form a disulfide bridge. We have investigated the stability of Aae Fd1 with (wild-type) and without (C87A variant) the disulfide bond, with respect to pH, thermal and chemical perturbation, and compared the results to those for the mesophilic [2Fe-2S] ferredoxin from spinach. Unfolding reactions of all three proteins are irreversible due to cluster decomposition in the unfolded state. Wild-type and C87A Aae Fd1 proteins are extremely stable: unfolding at 20 degrees C requires high concentrations of the chemical denaturant and long incubation times. Moreover, their thermal-unfolding midpoints are 40-50 degrees higher than that for spinach ferredoxin (pH 7). The stability of the Aae Fd1 protein is significantly lower at pH 2.5 than pH 7 and 10, suggesting that ionic interactions play a role in structural integrity. Interestingly, the iron-sulfur cluster in C87A Aae Fd1 rearranges into a transient species with absorption bands at 520 and 610 nm, presumably a linear three-iron cluster, in the high-pH unfolded state.     

Calix[8]arene-mediated self-assembly of tetrapeptide H-Leu-Leu-Ile-Leu-OMe

Conformational analysis of peptide 1, H-Leu-Leu-Ile-Leu-OMe on complexing with macro cycle calix[8]arene has been carried out using (1)H-NMR and FTIR spectroscopic techniques. Stoichiometry of the complex formed in the 1:8 ratio was evidenced by a Job plot. NMR studies of the above peptide show a marked downfield shift and an increase in (3)J values for NH resonances on complexing with calix[8]arene. The characteristic NOE connectivity between N(i+1)H and C(ialpha)H confirm beta-sheet conformation in the complexed state. Both (1)H-NMR and FTIR results indicate that the alpha-amino group of Leu I is proximal to the macrocycle and is involved in hydrogen bond formation with phenolic hydrogen atom of the calix[8]arene. This suggests that calix[8]arene provides a suitable platform for peptide 1 to self-assemble in a parallel beta-sheet conformation. The nature of calix[8]arene interaction with peptide 1 has been studied using dynamic NMR studies, which concludes that a bifurcated hydrogen bonding interaction exists in the molecular interfaces of the assembly.     

Morphological transforming activity and metabolism of cyclopenta-fused isomers of benz[a]anthracene in mammalian cells

4 isomeric cyclopenta-derivatives of benz[e]anthracene (benz[a]aceanthrylene, benz[j]aceanthrylene, benz[l]aceanthrylene, and benz[k]acephenanthrylene) were examined for their ability to morphologically transform C3H10T1/2CL8 mouse-embryo fibroblasts. All of these polycyclic aromatic hydrocarbons studied except benz[k]acephenanthrylene transformed C3H10T1/2CL8 cells to both type II and type III foci in a concentration-dependent fashion. Benz[j]aceanthrylene was the most active, equivalent in activity to benzo[a]pyrene on a molar basis, in producing dishes of cells with transformed foci (94% at 1.0 microgram/ml). Benz[e]aceanthrylene, and benz[l]aceanthrylene produced 58% and 85% of the dishes with foci respectively at 10 micrograms/ml. Metabolism studies with [3H]benz[j]aceanthrylene in C3H10T1/2CL8 cells in which unconjugated, glucuronic acid conjugated, and sulfate conjugated metabolites were measured indicated that the dihydrodiol precursor to the bay-region diol-epoxide, 9,10-dihydroxy-9,10-dihydrobenz[j]aceanthrylene, was the major dihydrodiol formed (55%). Smaller quantities of the cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[j]aceanthrylene (14%), and the k-region dihydrodiol, 11,12-dihydroxy-11,12-dihydrobenz[j]aceanthrylene (5%) were also formed. Similar studies with [14C]benz[l]aceanthrylene indicated that the k-region dihydrodiol, 7,8-dihydroxy-7,8-dihydrobenz[l]aceanthrylene was the major metabolite formed (45%). The cyclopenta-ring dihydrodiol, 1,2-dihydroxy-1,2-dihydrobenz[l]aceanthrylene and 4,5-dihydroxy-4,5-dihydrobenz[l]aceanthrylene were formed in minor amounts (less than 6%). Therefore, metabolism at the cyclopenta-ring of B(j)A and B(l)A is a minor pathway in C3H10T1/2CL8 cells in contrast to previously reported studies with cyclopenta[cd]pyrene in which the cyclopenta-ring dihydrodiol was the major metabolite. These results suggest that routes of metabolic activation other than oxidation at the cyclopenta-ring such as bay region or k-region activation may play an important role with these unique polycyclic aromatic hydrocarbons in C3H10T1/2CL8 cells.     

Reversible, positive cooperative interaction of 11 beta-chloromethyl-[3H]estradiol-17 beta with the calf uterine estrogen receptor

The binding of 11 beta-chloromethyl-[3H]estradiol-17 beta [3H]CME2) with the calf uterine estrogen receptor was investigated. The equilibrium binding analysis indicated a positive cooperative interaction yielding curvilinear Scatchard plots and Hill coefficients of 1.4-1.5. This positive cooperative interaction of [3H]CME2 was indistinguishable from the typical cooperative interaction of [3H]estradiol with the receptor. The apparent relative association constant and the relative binding affinity of CME2 for the estrogen receptor measured by competitive binding assay were 146 and 184%, respectively. The dissociation kinetics of [3H]CME2 from the receptor was biphasic, composed of a fast dissociating component (15%, t1/2 = 4 min at 0 degrees C; 9%, t1/2 = 4 min at 28 degrees C) and a slow dissociating component (85%, t1/2 greater than 50 h at 0 degrees C; 91%, t1/2 greater than 50 h at 28 degrees C). The dissociation kinetics of [3H]estradiol was also biphasic: the t1/2 of the fast dissociating component was 4 min at 0 and 28 degrees C and approximately 200 min for the slow dissociating component at both temperatures. The fraction of the slow [3H]estradiol dissociating component increased from 56 to 92% upon warming. Ethanol extraction and trichloroacetic acid treatment proved that the binding of [3H]CME2 is fully reversible. The unusual dissociation kinetics and the binding mechanism of CME2 are discussed.     

Preparation and characterization of oligonucleotides containing S-[2-(N7-guanyl)ethyl]glutathione.

S-[2-(N7-Guanyl)ethyl]glutathione is the major adduct derived from modification of DNA with 1,2-dibromoethane in biological systems and is postulated to be a mutagenic lesion [Humphreys, W. G., Kim, D.-H., Cmarik, J. L., Shimada, T., & Guengerich, F. P. (1990) Biochemistry 29, 10342-10350]. Oligonucleotides containing this modified base were prepared by treatment of oligonucleotides with S-(2-chloroethyl)glutathione and purified by chromatography. The self-complementary oligonucleotide d(ATGCAT), when thus modified at the single guanine, appeared to associate with itself as judged by UV measurements, but CD and NMR measurements indicated a lack of hybridization, with a decrease in the melting temperature of greater than 10 degrees C. The same lack of self-association was noted when d(ATGCAT) was modified to contain an N-acetyl-S-[2-(N7-guanyl)ethyl]cysteine methyl ester moiety. The oligomer d-(C1A2T3G4C5C6T7) was modified to contain a single S-[2-(N7-guanyl)ethyl]glutathione moiety at the central position, and UV, CD, and 1H NMR studies indicated that this oligomer hybridized to its normal complement d(A8G9G10C11A12T13G14), although the binding was considerably weakened by adduction (imino proton NMR spectroscopy in the presence of H2O indicated that the hydrogen bond signals seen in the oligomer were all broadened upon modification). All proton resonances were identified using two-dimensional 1H NMR spectroscopy. Adduct formation affected the chemical shifts of the base and 1', 2', and 2" protons of T3 and C5, the 2" proton of C6, and the 8 and 1' protons of C11, while little effect was observed on other protons. No cross-peaks were detected between the glutathione and oligomer moieties in two-dimensional nuclear Overhauser enhanced NMR studies. These results suggest that a rather local structural perturbation occurs in the DNA oligomer upon modification and that the glutathione moiety appears to be relatively unperturbed by its placement in the duplex. When the cytosine in the normal d(AGGCATG) complement to d-(CATGCCT) was changed to each of the other three potential bases at the central position, no hybridization with the oligomer d(CATGCCT) containing S-[2-(N7-guanyl)ethyl]glutathione was detected. We conclude that these N7-guanyl derivatives destabilize hybridization and that bases other than cytosine do not appear to show preferential thermodynamic bonding to these adducts, at least in the sequences examined to date.(ABSTRACT TRUNCATED AT 400 WORDS)