Active site labeling of the gentamicin resistance enzyme AAC(6')-APH(2") by the lipid kinase inhibitor wortmannin

BACKGROUND: Aminoglycoside antibiotic resistance is largely the result of the production of enzymes that covalently modify the drugs including kinases (APHs) with structural and functional similarity to protein and lipid kinases. One of the most important aminoglycoside resistance enzymes is AAC(6')-APH(2"), a bifunctional enzyme with both aminoglycoside acetyltransferase and kinase activities. Knowledge of enzyme active site structure is important in deciphering the molecular mechanism of antibiotic resistance and here we explored active site labeling techniques to study AAC(6')-APH(2") structure and function. RESULTS: AAC(6')-APH(2") was irreversibly inactivated by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, through the covalent modification of a conserved lysine in the ATP binding pocket. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine, an electrophilic ATP analogue and known inactivator of other APH enzymes such as APH(3')-IIIa, did not inactivate AAC(6')-APH(2"), and reciprocally, wortmannin did not inactivate APH(3')-IIIa. CONCLUSIONS: These distinct active site label sensitivities point to important differences in aminoglycoside kinase active site structures and suggest that design of broad range, ATP binding site-directed inhibitors against APHs will be difficult. Nonetheless, given the sensitivity of APH enzymes to both protein and lipid kinase inhibitors, potent lead inhibitors of this important resistance enzyme are likely to be found among the libraries of compounds directed against other pharmacologically important kinases.     

Tethered bisubstrate derivatives as probes for mechanism and as inhibitors of aminoglycoside 3'-phosphotransferases

Aminoglycoside 3'-phosphotransferases [APH(3')s] phosphorylate aminoglycoside antibiotics, a reaction that inactivates the antibiotics. These enzymes are the primary cause of resistance to aminoglycosides in bacteria. APH(3')-Ia operates by a random-equilibrium BiBi mechanism, whereas APH(3')-IIIa catalyzes its reaction by the Theorell-Chance mechanism, a form of ordered BiBi mechanism. Hence, both substrates have to be present in the active site prior to the transfer of phosphate by both mechanisms. Four bisubstrate analogues, compounds 1-4, were designed and synthesized as inhibitors for APH(3')s. These compounds are made of adenosine linked covalently to the 3'-hydroxyl of neamine (an aminoglycoside) via all-methylene tethers of 5-8 carbons. The K(i) values measured for these compounds indicated that affinities of APH(3')-Ia and APH(3')-IIa for compounds 2 and 3 (six- and seven-carbon tethers, respectively) were the best, and the inhibition constants for the two were comparable.     

Dissection of aminoglycoside-enzyme interactions: a calorimetric and NMR study of neomycin B binding to the aminoglycoside phosphotransferase(3')-IIIa

In this work, for the first time, we report pKa values of the amino functions in a target-bound aminoglycoside antibiotic, which permitted dissection of the thermodynamic properties of an enzyme-aminoglycoside complex. Uniformly enriched 15N-neomycin was isolated from cultures of Streptomyces fradiae and used to study its binding to the aminoglycoside phosphotransferase(3')-IIIa (APH) by 15N NMR spectroscopy. 15N NMR studies showed that binding of neomycin to APH causes upshifts of approximately 1 pKa unit for the amines N2' and N2' ' while N6' experienced a 0.3 pKa unit shift. The pKa of N6' ' remained unaltered, and resonances of N1 and N3 showed significant broadening upon binding to the enzyme. The binding-linked protonation and pH dependence of the association constant (Kb) for the enzyme-aminoglycoside complex was determined by isothermal titration calorimetry. The enthalpy of binding became more favorable (negative) with increasing pH. At high pH, binding-linked protonation was attributable mostly to the amino functions of neomycin; however, at neutral pH, functional groups of the enzyme, possibly remote from the active site, also underwent protonation/deprotonation upon formation of the binary enzyme-neomycin complex. The Kb for the enzyme-neomycin complex showed a complicated dependence on pH, indicating that multiple interactions may affect the affinity of the ligand to the enzyme and altered conditions, such as pH, may favor one or another. This work highlights the importance of determining thermodynamic parameters of aminoglycoside-target interactions under different conditions before making attributions to specific sites and their effects on these global parameters.     

咪唑类离子液体与酪氨酸相互作用及机理的密度泛函理论研究

Ionic liquids (ILs) are thermally and chemically stable and have adjustable structures, which gives them the potential to be used as green, efficient biomolecular solvents. Given the critical role of ILs in dissolving biomolecules, the mechanism of interaction between them deserves further study. Herein, density functional theory (DFT) calculations, using the SMD implicit water solvent model, were employed to study the interaction and mechanism between a hydrophobic zwitterionic amino acid (Tyr) and a series of imidazolium ILs with different alkyl chain lengths and methylation sites. The contributions of hydrogen bonding (H-bonding), electrostatic effects, induction, and dispersion to the intermolecular interactions were determined by combining the symmetry-adapted perturbation theory (SAPT), the atoms in molecules (AIM) theory, and reduced density gradient (RDG) analysis. The results indicate that the H-bonding between the IL cation and Tyr is stronger than that between the IL anion and Tyr; however, the binding between either ion and Tyr is dominated by electrostatic effects. By contrast, the difference between the induction and dispersion forces is small when methylation occurs on the C2 site of the imidazolium cation; whereas, it is significantly large when methylation takes place on the N3 site. This is rationalized by the interaction patterns that vary based on the methylation site. H-bonding and π⁺-π stacking interactions between the imidazole and benzene rings are dominant during C2-methylation, while H-bonding and C_Alkyl-H…π interactions between the alkyl chain and benzene ring are dominant during N3-methylation. Increasing the side alkyl chain length has different effects on the interaction energy to cations with different methylation sites. During N3-methylation, when the side alkyl chain length increases from 4 to 12, there are significant van der Waals interactions between the Tyr benzene and the side alkyl chain. However, these van der Waals interactions are inapparent when methylation takes place on the C2 site. Finally, the synergetic effect of the H-bonding and the interaction between the benzene and the side alkyl chain for C2-methylation is greater than the H-bonding and the interaction between the imidazole and benzene rings for N3-methylation, when the side alkyl chain length n > 9. Therefore, the interaction strength and mechanism in these imidazolium-Tyr complexes can be regulated by changing the methylation site and the side alkyl chain length of the cation. Further study of ion-pair and Tyr reveals that the change tendency of the interaction energy of IL-Tyr systems is consistent with that of cation-Tyr cases, and the ion pair further stabilizes the binding with Tyr. These results illustrate the interaction mechanism of IL-Tyr systems and provide a novel strategy for the design and screening of functional ILs for amino acid extraction and separation in the future.     

Aromatic interaction profile to understand the molecular basis of raltegravir resistance

Integrase (IN) is the enzyme of human immunodeficiency virus (HIV) which inserts the viral DNA (vDNA) into the host genome for successful viral replication leading to the infection. However, the chemical basis of HIV IN catalysis is speculative due to lack of complete co-crystal structure. Using the recently published prototype foamy virus IN crystal structure, we developed a model structure of HIV IN showing interaction of vDNA, the metal (Mg²⁺) cofactor, and raltegravir (RLT) in the active site. Molecular docking and dynamics simulations studies showed that RLT uses it core central ring with diketo motif for Mg²⁺ chelation and bridge interaction with DDE motif. The triple arene interactions mediated by RLT with neighboring molecular motifs (Y143, cytosine, and adenine) is maintained during long simulation in wild type (WT). The fluorobenzyl and oxadiazole moieties of RLT forms aromatic stacking with cytosine base (head stacking) aromatic side chain of Y143 (tail stacking), respectively, while central ring further establishes aromatic stacking with distorted adenine base of vDNA (central stacking). The novel triple stacking systems were further explored to understand the molecular basis of drug resistance by molecular simulation. The in silico mutation (N155H, Q148H, and Q148H + G140S) and simulation studies elucidated the structural mechanism of resistance to RLT. The simulation studies provided the molecular basis for interdependency observed for the primary and secondary (Q148H and G140S) mutations and also explained the mechanism of viral fitness regain. Our study reveals that triple stacking and its consequence in terms of VdW energetic profile acts as a critical point to understand the drug-resistance. Here, we demonstrate that the root mean square deviation of centroid system (aromatic stacking) can be used as a major determinant of RLT binding toward the fold resistance. This is first kind of report, which discloses a strategy to explore the molecular level of drug resistance profile using aromatic interactions.     

Chemical shifts for the unusual DNA structure in Pf1 bacteriophage from dynamic-nuclear-polarization-enhanced solid-state NMR spectroscopy

Solid-state NMR spectra, including dynamic nuclear polarization enhanced 400 MHz spectra acquired at 100 K, as well as non-DNP spectra at a variety of field strengths and at temperatures in the range 213-243 K, have allowed the assignment of the (13)C and (15)N resonances of the unusual DNA structure in the Pf1 virion. The (13)C chemical shifts of C3' and C5', considered to be key reporters of deoxyribose conformation, fall near or beyond the edges of their respective ranges in available databases. The (13)C and (15)N chemical shifts of the DNA bases have above-average values for AC4, AC5, CC5, TC2, and TC5, and below average values for AC8, GC8, and GN2, pointing to an absence of Watson-Crick hydrogen bonding, yet the presence of some type of aromatic ring interaction. Crosspeaks between Tyr40 of the coat protein and several DNA atoms suggest that Tyr40 is involved in this ring interaction. In addition, these crosspeak resonances and several deoxyribose resonances are multiply split, presumably through the effects of ordered but differing interactions between capsid protein subunits and each type of nucleotide in each of the two DNA strands. Overall, these observations characterize and support the DNA model proposed by Liu and Day and refined by Tsuboi et al., which calls for the most highly stretched and twisted naturally occurring DNA yet encountered.     

Structural and binding study of modified siRNAs with the Argonaute 2 PAZ domain by NMR spectroscopy

By using high-resolution NMR spectroscopy, the structures of a natural short interfering RNA (siRNA) and of several altritol nucleic acid (ANA)-modified siRNAs were determined. The interaction of modified siRNAs with the PAZ domain of the Argonaute 2 protein of Drosophila melanogaster was also studied. The structures show that the modified siRNA duplexes (ANA/RNA) adopt a geometry very similar to the naturally occurring A-type siRNA duplex. All ribose residues, except for the 3' overhang, show 3'-endo conformation. The six-membered altritol sugar in ANA occurs in a chair conformation with the nucleobase in an axial position. In all siRNA duplexes, two overhanging nucleotides at the 3' end enhance the stability of the first neighboring base pair by a stacking interaction. The first overhanging nucleotide has a rather fixed position, whereas the second overhanging nucleotide shows larger flexibility. NMR binding studies of the PAZ domain with ANA-modified siRNAs demonstrate that modifications in the double-stranded region of the antisense strand have some small effects on the binding affinity as compared with the unmodified siRNA. Modification of the 3' overhang with thymidine (dTdT) residues shows a sixfold increase in the binding affinity compared with the unmodified siRNA (relative binding affinity of 17% compared with dTdT-modified overhang), whereas modification of the 3' overhang with ANA largely decreases the binding affinity.     

Minimalist protein design: a beta-hairpin peptide that binds ssDNA

A 28-residue beta-hairpin dimer (WKWK)2 with two Trp and two Lys residues on one face of each beta-sheet was shown to form a complex with single-stranded oligonucleotides at low micromolar concentrations. Each beta-hairpin of the dimer contains a cross-strand Trp-Trp pair in a diagonal orientation which has previously been shown to create a cleft for the intercalation of aromatic guests such as adenine (J. Am. Chem. Soc. 2003, 125, 9580). The beta-hairpin dimer binds 5-residue ssDNA sequences 5'-AAAAA-3', 5'-TTTTT-3', and 5'-CCCCC-3' in water with dissociation constants in the range of 12-30 muM. A weak energetic preference for binding to sequence 5'-AAAAA-3' was observed, which is believed to result from stronger stacking interactions between Trp and the adenine base. The interaction of 5'-AAAAA-3' with the Lys and Trp residues of the peptide was evident by NMR, and a 1:1 association was demonstrated. The recognition of an 11-residue ssDNA sequence occurred with a dissociation constant of 3 muM under near-physiological ionic strength and pH, demonstrating that the beta-hairpin dimer binds ssDNA as strongly as many naturally occurring proteins. The salt dependence of the interaction of the 11-residue oligonucleotide with the peptide dimer indicates that Trp-nucleobase stacking interactions contribute about -4 kcal/mol to recognition, which is much greater than the contribution of nonionic interactions in unstructured peptides containing Trp. Moreover, recognition of the ssDNA demonstrated reduced salt dependence relative to the corresponding duplex, resulting in selectivity for ssDNA under high salt conditions. Peptide (WKWK)2 is a relevant mimic of OB-fold (oligonucleotide/oligosaccharide-binding) proteins which bind ssDNA on the surface of a beta-sheet.     

Tyrosine Residues 232 and 401 Play a Critical Role in the Binding of the Cofactor FAD of Acyl-coA Oxidase

Acyl-coA oxidase (ACO) is an important flavoenzyme responsible for the first step of peroxisomal fatty acid β-oxidation. In this study, the roles of Tyr232 and Tyr401 in flavin adenine dinucleotide (FAD) binding and enzyme catalysis of ACO were explored using site-directed mutagenesis. For mutant proteins, different levels of activity loss were observed. Wavelength scanning of Y232 and Y401 mutant proteins indicated that there is no FAD binding in Y401S and Y401G mutant ACO. Structure analysis indicated that the phenolic hydroxyl and benzene ring of the side chain could stabilize FAD binding through hydrogen bonds network and hydrophobic pocket formation. These results indicated that these two tyrosine residues play a critical role in the FAD binding of ACO.     

Differential effects of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride on the recognition and catalysis in ATP hydrolysis

The supramolecular interactions of Mg(ii) and N(alpha)-4-tosyl-l-arginine methyl ester hydrochloride (TAME) with ATP have been investigated using (1)H and (31)P NMR spectra. Furthermore, the hydrolysis of ATP catalyzed by Mg(ii) and TAME has been studied at 60 degrees C and pH 7 using (31)P NMR spectra. In the Mg(ii)-ATP-TAME ternary system, the binding interaction of Mg(2+) with ATP involves not only N1 and N7 in the adenine ring but also beta- and gamma-phosphate of ATP. The binding forces are mainly electrostatic interaction and cation (Mg(2+))-pi interaction. The guanidinium group and the aromatic ring of TAME interacts with ATP by beta and gamma phosphate and the adenine ring of ATP. The binding forces are mainly electrostatic interactions and pi-pi stacking. A significant difference between the binary and the ternary system indicates that TAME is essential to the stablization of the intermediate. Kinetic studies show that the hydrolysis rate constant of ATP is 2.16 x 10(-2) h(-1) at pH 7 in the Mg(ii)-TAME-ATP ternary system. The Mg(ii) ion and TAME can accelerate the ATP hydrolysis process. A possible mechanism has been proposed that the hydrolysis occurs through an addition-elimination, in which the phosphoramidate intermediate was observed at 3.21 ppm in the (31)P NMR of the ternary system. These results provide further information concerning the effect of the key amino acid residue and metal ions as cofactors of ATPase on ATP synthesis/hydrolysis at the molecular level.     

Role of nitrogen atom in aromatic stacking

Aromatic stacking of 9,9'-(alpha,omega-alkanediyl)bis[adenine] (1), 1,1'-(alpha,omega-alkanediyl)bis[benzimidazole] (2), and 9-[omega-(benzimidazol-1-yl)alkyl]adenine (3) were studied at low concentrations of these compounds by means of UV and NMR spectroscopies. The UV hypochromic effect at T degrees C was determined as the ratio of the integration strength B at T degrees C (T = 27, 40, and 50) to that at 60 degrees C. The UV hypochromic effects of 1 and 3 were remarkable in water, suggesting a formation of intramolecular aromatic stacking, while the UV data of 2 did not present unambiguous evidence supporting aromatic stacking. A difference of chemical shift of each aromatic ring proton between 27 and 80 degrees C, that is Deltadelta = delta(80 degrees C) - delta(27 degrees C), was given as an indication of the aromatic stacking in the NMR study. On the basis the data of Deltadelta, 1 and 3 were stabilized by a stacking interaction in the buffer solution at pD 7.0 but not in the organic solvents. On the other hand, the NMR data did not indicate the formation of aromatic stacking of 2 either in the organic solvents or in the aqueous solution. The thermodynamic parameters of the intramolecular aromatic stacking of 3 were determined by means of NMR spectroscopy.     

Characterization of nucleobase-amino acid stacking interactions utilized by a DNA repair enzyme

The present work characterizes the gas-phase stacking interactions between four aromatic amino acid residues (histidine, phenylalanine, tyrosine, and tryptophan) and adenine or 3-methyladenine due to the proposed utilization of these interactions by enzymes that repair DNA alkylation damage. The MP2 potential energy surfaces of the stacked dimers are considered as a function of four variables (vertical displacement, angle of rotation, horizontal displacement, and tilt angle) using a variety of basis sets. It is found that the maximum stacking interaction energy decreases with the amino acid according to TRP > TYR approximately HIS > PHE for both nucleobases. However, the magnitude of the stacking interaction significantly increases upon alkylation (by 50-115%). Comparison of the stacking energies calculated using our surface scans to those estimated from experimental crystal structures indicates that the stacking interactions within the active site of 3-methyladenine DNA glycosylase can account for 65-75% of the maximum possible stacking interaction between the relevant molecules. The decrease in stacking in the crystal structure arises due to significant differences in the relative orientations of the nucleobase and amino acid. Nevertheless, alkylation is found to significantly increase the stacking energy when the crystal structure geometries are considered. Our calculations provide computational support for suggestions that alkylation enhances the stacking interactions within the active site of DNA repair enzymes, and they give a measure of the magnitude of this enhancement. Our results suggest that alkylation likely plays a more important role in substrate identification and removal than the nature of the aromatic amino acid that interacts with the substrate via stacking interactions.     

Three-dimensional quantitative structure-activity relationship of nucleosides acting at the A3 adenosine receptor: analysis of binding and relative efficacy

The binding affinity and relative maximal efficacy of human A3 adenosine receptor (AR) agonists were each subjected to ligand-based three-dimensional quantitative structure-activity relationship analysis. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) used as training sets a series of 91 structurally diverse adenosine analogues with modifications at the N6 and C2 positions of the adenine ring and at the 3', 4', and 5' positions of the ribose moiety. The CoMFA and CoMSIA models yielded significant cross-validated q2 values of 0.53 (r2 = 0.92) and 0.59 (r2 = 0.92), respectively, and were further validated by an external test set (25 adenosine derivatives), resulting in the best predictive r2 values of 0.84 and 0.70 in each model. Both the CoMFA and the CoMSIA maps for steric or hydrophobic, electrostatic, and hydrogen-bonding interactions well reflected the nature of the putative binding site previously obtained by molecular docking. A conformationally restricted bulky group at the N6 or C2 position of the adenine ring and a hydrophilic and/or H-bonding group at the 5' position were predicted to increase A3AR binding affinity. A small hydrophobic group at N6 promotes receptor activation. A hydrophilic and hydrogen-bonding moiety at the 5' position appears to contribute to the receptor activation process, associated with the conformational change of transmembrane domains 5, 6, and 7. The 3D-CoMFA/CoMSIA model correlates well with previous receptor-docking results, current data of A3AR agonists, and the successful conversion of the A3AR agonist into antagonists by substitution (at N6) or conformational constraint (at 5'-N-methyluronamide).     

Inhibition of mitochondrial respiratory chain by arylthiolated 2,3-ethylenedioxy-1,4-benzoquinones

A series of arylthiolated 2,3-ethylenedioxy-1,4-benzoquinones as a coenzyme Q (CoQ) antagonist was tested for inhibition of succinate oxidase and reduced nicotinamide adenine dinucleotide (NADH) oxidase systems in the mitochondrial respiratory chain. The following characteristics were revealed: (1) 2,3-ethylenedioxy, 5-arylthio and 5,6-diarylthio groups were confirmed to be favorable for inhibition of both systems; (2) these analogs were more effective in the succinate oxidase system than in the NADH oxidase system; (3) 4' substituents on the benzene side ring had little effect on inhibitory activity; (4) the acting sites of these analogs had no strict stereospecificity. The reduced minus oxidized difference spectra revealed that these analogs inhibited the succinate oxidase system at the site between succinate and CoQ, and the NADH oxidase system at the site after cytochrome a + a3, suggesting these analogs might act as antagonists of CoQ in the succinate oxidase system. However, 5-(4'-nitrophenylthio)-2,3-ethylenedioxy-1,4-benzoquinone (If) strongly inhibited only the succinate oxidase system at the site after cytochrome a + a3.     

Structures and Stabilities of Ternary Copper(II) Complexes with 3,5-Diiodo-L-tyrosinate. Weak Interactions Involving Iodo Groups

Structures and stabilities of the ternary copper(II) complexes Cu(DA)(AA), where AA refers to 3,5-diiodo-L-tyrosinate (I(2)tyr) or L-tyrosinate (Tyr) and DA refers to 1,10-phenanthroline (phen), 2,2'-bipyridine (bpy), 2-(aminomethyl)pyridine (ampy), histamine (hista), or ethylenediamine (en), have been investigated by potentiometric, spectroscopic, and X-ray diffraction methods. The stability constants have been determined by potentiometric titrations at 25 degrees C and ionic strength I = 0.1 M (KNO(3)). The equilibrium constants K for a hypothetical equilibrium, Cu(DA)(Ala) + Cu(en)(AA) Cu(DA)(AA) + Cu(en)(Ala) where Ala refers to L-alanine, have been calculated from the determined overall stability constants of the ternary complexes for estimating the stability enhancement due to the stacking interaction between the aromatic rings in Cu(DA)(AA). Large positive log K values have been obtained for the Cu(DA)(I(2)tyrOH) and Cu(DA)(I(2)tyrO(-)) systems (DA = phen or bpy, OH and O(-) refer to the protonated and deprotonated forms of the phenol moiety, respectively), indicating that the complexes are stabilized by effective stacking. Differences between the log K values for Cu(DA)(I(2)tyr) and Cu(DA)(Tyr) systems indicate that the iodine substituents greatly contribute to the stability enhancement. A distinct circular dichroism (CD) magnitude anomaly was also observed for the systems with large log K value, supporting the existence of the stacking interaction in Cu(DA)(AA). Two complexes, [Cu(bpy)(I(2)tyrO(-))(H(2)O)].2H(2)O (1) and [Cu(bpy)(I(2)tyrOH)(NO(3))].CH(3)OH (2), have been isolated as crystals and analyzed by the X-ray diffraction method. Both 1 and 2 crystallized in the orthorhombic space group P2(1)2(1)2(1) with four molecules in a unit cell of dimensions a = 9.2339(4), b = 16.9230(8), and c = 14.8584(5) ? for complex 1, and a = 11.2240(8), b = 11.715(1), and c = 17.966(2) ? for complex 2. The central Cu(II) ion for both complexes has a similar distorted five-coordinate square-pyramidal geometry with the equatorial positions occupied by the two nitrogen atoms of bpy and the nitrogen and oxygen atoms of I(2)tyr, and the apical position is occupied by a water molecule (for 1) or a nitrate ion (for 2). The opposite site to the axial water or nitrate oxygen atom is intramolecularly occupied by the side chain aromatic ring, which is approximately parallel to the copper coordination plane with the average spacing of 3.31 or 3.30 ? for complex 1 or 2, respectively, directly exhibiting the effective stacking interaction between the aromatic rings in the solid state. Distances between the iodine and one of the pyridine rings of bpy (3.79 ? for 1 and 3.56 ? for 2) are shorter than the van der Waals distance (3.85 ?), implying that the iodine substituent may be involved in a weak bonding interaction with the pyridine ring. Effects of the iodine substituents on the stacking interactions between the diiodophenol side ring and the coordinated aromatic diamine and their possible biological relevance have been discussed.     

带电组氨酸侧链与DNA碱基间非键作用强度的理论研究

采用MP2方法和6-31+G(d,p)基组优化得到了带有一个正电荷的组氨酸侧链与4个DNA碱基间形成的18个氢键复合物的气相稳定结构, 从文献中获取了组氨酸侧链与DNA碱基间形成的12个堆积和T型复合物的气相稳定结构, 使用包含基组重叠误差(BSSE)校正的MP2方法和aug-cc-pVTZ基组及密度泛函理论M06-2X-D3方法和aug-cc-pVDZ基组计算了这些复合物的结合能. 研究结果表明, 包含BSSE校正的M06-2X-D3方法和aug-cc-pVDZ基组能够给出较准确的结合能; 气相条件下, 组氨酸侧链与同种DNA碱基间的离子氢键作用明显强于堆积作用和T型作用, 组氨酸侧链最易通过离子氢键与胞嘧啶C和鸟嘌呤G作用形成氢键复合物, 组氨酸与胞嘧啶C和鸟嘌呤G间的T型作用强于与腺嘌呤A和胸腺嘧啶T间的离子氢键作用; 水相条件下, 组氨酸侧链与同种DNA碱基间的离子氢键作用仍明显强于堆积作用和T型作用, 组氨酸侧链更易与胞嘧啶C和鸟嘌呤G相互作用形成氢键复合物, 但是最强的组氨酸侧链与胞嘧啶C间的T型作用明显弱于与腺嘌呤A和胸腺嘧啶T间的离子氢键作用, 说明水相条件下组氨酸侧链与DNA碱基间主要通过离子氢键作用形成氢键复合物.     

Interactions and self-organization of nucleic bases,nucleotides, and polynucleotides into ordered structures. Effect of ionization and solvent salt composition

This paper summarizes our findings on the effects of protons and neutral salt ions on intermolecular interactions and the self-organization of nucleotide systems in aqueous media, both at monomer and polymer levels. To gain quantitative information about these processes, methods were developed to determine the thermodynamic parameters of monomer association from the data obtained by various experimental techniques (NMR , UV spectroscopy, and spin labeling) and to estimate the individual contributions of base stacking and H-bonding in ordered structures of polynucleotides and their complexes. The main results obtained using these methods are as follows. (i) A difference was discovered in the effects of positive and negative charges on the stacking interaction between ionized and neutral molecules of nucleic heterocycles. Protonation strongly decreases the heterocycle ability to form ordered stacks whereas deprotonation slightly affects stacking. (ii) Base–phosphate interaction, which has not yet really been investigated, was studied. This interaction, along with base stacking, governs nucleotide association in water media. It appears upon protonation of the base moiety and increases with decreasing ionic strength. (iii) Base stacking was found to become stronger under the action of salt anions stabilizing the water structure and it became weaker under the action of destabilizing salt anions, both in monomer and polymer systems, which is indicative of an indirect mechanism of the anionic effects. It is essential that small salt ion additives acting by such a “distance” mechanism can affect the formation of ordered structures in nucleotide systems as strong as the direct interaction of the bases with protons. (iv) The results obtained suggest that an increase in solvent entropy upon the self-organization of nucleotide systems in aqueous media may be an important factor promoting these processes. (v) As follows from our data, specific weakening or strengthening of intermolecular interactions by protons and salt ions at small, physiological-range changes in pH or solvent salt composition seems to be an effective regulatory mechanism for the functioning of nucleotide systems.     

Controlled aggregation of adenine by sugars: physicochemical studies, molecular modelling simulations of sugar-aromatic CH-pi stacking interactions, and biological significance

CH-Pi stacking interactions between carbohydrates and aromatic compounds play a central role in biomolecular recognition, especially in lectin-sugar and protein-glycolipid systems. In the present study, we have measured the solubility of the sparingly soluble aromatic base adenine in presence of various saccharides as an approach to investigate the interaction between adenine and sugars. Above 82.5 mM, adenine solutions gradually formed a crystalline precipitate which could be quantified by spectrophotometric turbidity measurements. Precipitation of adenine was increased by salts (NaCl and NaF) whereas it was prevented by DMSO, in agreement with the involvement of hydrophobic interactions (pi-pi stacking) in the vertical stacking of adenine molecules. Several monosaccharides and disaccharides were found to increase adenine solubility, with the following order: D-galactose = D-lactose > D-sucrose > D-glucose = D-maltose > D-ribose > D-fructose. Molecular mechanics simulations indicated that the potent cosolvent effect of beta-D-galactopyranose was probably mediated by CH-pi stacking interactions between its apolar surface and the aromatic structure of adenine. The polar OH groups of the sugars interacted with surrounding water molecules, ensuring the solubility of sugar-adenine complexes. In contrast, beta-D-fructofuranose, which has two polar faces, did not stack onto adenine and had a weak cosolvent effect. CH-pi stacking interactions were also demonstrated between 6-methylpurine and the sugar head group of glycolipids (glucosyl-, galactosyl- and lactosylceramide) but not with the charged head group of phosphatidylinositol-4,5-diphosphate. These data indicate that galactose-containing molecules have a high stacking propensity for aromatic compounds such as adenine, due to the specific structure of the galactose cycle.     

1~HNMR STUDY OF INTRAMOLECULAR STACKING IN TERNARY PALLADIUM（Ⅱ） COMPLEXES INVOLVING ADENOSLNE 5＇－TRIPHOSPHATE AND LIGANDS CONTAINING OXYGEN AND／OR NITROGEN AS DONOR ATOMS

１￣ＨＮＭＲＳＴＵＤＹＯＦＩＮＴＲＡＭＯＬＥＣＵＬＡＲＳＴＡＣＫＩＮＧＩＮＴＥＲＮＡＲＹＰＡＬＬＡＤＩＵＭ（Ⅱ）ＣＯＭＰＬＥＸＥＳＩＮＶＯＬＶＩＮＧＡＤＥＮＯＳＬＮＥ５＇－ＴＲＩＰＨＯＳＰＨＡＴＥＡＮＤＬＩＧＡＮＤＳＣＯＮＴＡＩＮＩＮＧＯＸＹＧＥＮＡ...     

Anti versus syn opening of epoxides derived from 9-(3-deoxy-beta-D-glycero-pent-3-enofuranosyl)adenine with Me3Al: factors controlling the stereoselectivity

Upon epoxidation with dimethyldioxirane, the 2',5'-bis-O-silyl derivatives of 9-(3-deoxy-beta-D-glycero-pent-3-enofuranosyl)adenine gave the respective "3',4'-up" epoxides exclusively. Reaction between these epoxides and Me3Al was investigated in detail. It was found that the stereoselectivity of epoxide ring opening (anti versus syn) varied significantly upon changing the amount of Me3Al, the solvent, the O-silyl protecting group, and the reaction temperature. A possible reaction mechanism is proposed.