Rational design of peptide ligand for affinity chromatography of tissue-type plasminogen activator by the combination of docking and molecular dynamics simulations

Combined docking and molecular dynamics (MD) simulations are carried out for the rational design of affinity peptide ligand of tissue-type plasminogen activator (t-PA). Ten amino acids that have high affinity to three different regions of t-PA are identified by the amino acids location method on the basis of candidate pocket structure of t-PA. Then, 14 tetrapeptides are built and docked into the candidate pocket of t-PA. The absolute value of the D(score) calculated from the docking simulation is used to assess the affinity of a peptide for t-PA. Consequently, six tetrapeptides that have high D(score) values are selected and linked to a spacer arm of [NH(CH(2))(6)NH(2)] that is present on EAH Sepharose gel. The linked compounds are further evaluated by docking into the candidate pocket of t-PA. As a result, the tetrapeptide QDES with the highest D(score) value is selected. Molecular surface analysis with the MOLCAD program reveals that electrostatic interactions and hydrogen bonds (H-bonds) contribute to the affinity interactions between the tetrapeptide and t-PA. MD simulations indicate that QDES-t-PA complex keeps stable, and the distances between the carboxyl groups of Asp189, Gln192 and Asp194 and the charged amino group of glutamine change little. Moreover, all the nine H-bonds found in the docking simulation are confirmed by the MD simulations. It is also found that three water molecules act as bridges between the ligand and the protein pocket by hydrogen bonding. Finally, high binding affinity and specificity of the peptide ligand are confirmed by the purification of t-PA from crude porcine heart extract using the immobilized-ligand column for affinity chromatography.     

High‐Affinity Inhibitors of tRNA‐Guanine Transglycosylase Replacing the Function of a Structural Water Cluster

The tRNA‐modifying enzyme tRNA–guanine transglycosylase (TGT) is essential for the pathogenic mechanism of Shigella flexneri, the causing agent of the bacterial diarrheal disease shigellosis. Herein, the synthesis of a new class of rationally designed 6‐amino‐imidazo[4,5‐g]quinazolin‐8(7H)‐one‐ (lin‐benzoguanine) based inhibitors of TGT are reported. In order to accommodate a small hydrophobic crevice opening near the binding site of ribose‐34, 2‐aminoethyl substituents were introduced in position 4 of the heterocyclic scaffold. For this purpose, a synthetic sequence consisting of iodination, Suzuki cross‐coupling, hydroboration, Mitsunobu reaction, and Gabriel synthesis was employed, furnishing a primary amine that served as a common intermediate for the preparation of a series of derivatives. The resulting ligands displayed very low inhibition constants, down to K_i=2 nM . Substantial additional inhibitory potency is gained by interaction of terminal lipophilic groups attached to the substituent at position 4 with the hydrophobic crevice shaped by Val45 and Leu68. At the same time, the secondary ammonium center in the substituent displaces a cluster of water molecules, solvating the catalytic residues Asp102 and Asp280, without loss in binding affinity. In addition, a synthetic intermediate with an unusual 3,6,7,8,9,10‐hexahydroimidazo[4,5‐g][1,3]benzodiazepine core, as confirmed by X‐ray analysis, is reported.     

Replacement of Water Molecules in a Phosphate Binding Site by Furanoside‐Appended lin‐Benzoguanine Ligands of tRNA‐Guanine Transglycosylase (TGT)

The enzyme tRNA‐guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure‐based design for this enzyme is the filling of the polar ribose‐34 pocket. Herein, we describe a novel series of ligands consisting of furanoside‐appended lin‐benzoguanines. They were designed to replace a conserved water cluster and differ by the functional groups at C(2) and C(3) of the furanosyl moiety being either OH or OMe. The unfavorable desolvation of Asp102 and Asp280, which are located close to the ribose‐34 pocket, had a significant impact on binding affinity. While the enzyme has tRNA as its natural substrate, X‐ray co‐crystal structures revealed that the furanosyl moieties of the ligands are not accommodated in the tRNA ribose‐34 site, but at the location of the adjacent phosphate group. A remarkable similarity of the position of the oxygen atoms in these two structures suggests furanosides as a potential phosphate isoster.     

Thermodynamics of buried water clusters at a protein-ligand binding interface

The structure of the complex of cyclophilin A (CypA) with cyclosporin A (CsA, 1) shows a cluster of four water molecules buried at the binding interface, which is rearranged when CsA is replaced by (5-hydroxynorvaline)-2-cyclosporin (2). The thermodynamic contributions of each bound water molecule in the two complexes are explored with the inhomogeneous fluid solvation theory and molecular dynamics simulations. Water (WTR) 133 in complex 1 contributes little to the binding affinity, while WTR6 and 7 in complex 2 play an essential role in mediating protein-ligand binding with a hydrogen bond network. The calculations reveal that the rearrangement of the water molecules contributes favorably to the binding affinity, even though one of them is displaced going from ligand 1 to 2. Another favorable contribution comes from the larger protein-ligand interactions of ligand 2. However, these favorable contributions are not sufficient to overcome the unfavorable desolvation free energy change and the conformational entropy of the hydroxylpropyl group of ligand 2 in the complex, leading to a lower binding affinity of ligand 2. These physical insights may be useful in the development of improved scoring functions for binding affinity prediction.     

Migration of a cis-(NH3)2PtII moiety along two adenine nucleobases, from N1 to N6, is markedly facilitated by additional PtII entities coordinated to N7

Adenine acidification as a consequence of simultaneous PtII binding to N1 and N7 facilitates deprotonation of the exocyclic N(6)H2 group and permits PtII migration from N1 to N6 under mild conditions. Starting from the trinuclear complex cis-[(NH3)2Pt(N1-9-MeA-N7)2{Pt(NH3)3)}2]6+ (3), stepwise migration of cis-(NH3)2PtII takes place in the alkaline aqueous solution to give initially cis-[(NH3)2Pt(N1-9-MeA-N7)(N6-9-MeA--N7){Pt(NH3)3}2]5+ (4) and eventually cis-[(NH3)2Pt(N6-9-MeA--N7)2{Pt(NH3)3}2]4+ (5) (with 9-MeA = neutral 9-methyladenine, 9-MeA- = 9-methyl-adenine monoanion, deprotonated at N6). The migration process has been studied by 1H NMR spectroscopy, and relevant acid-base equilibria have been determined. 5 has been crystallized as its nitrate salt and has been characterized by X-ray crystallography. The precursor of 3, [(NH3)3Pt (9-MeA-N7)]Cl2.2H2O (2) has likewise been studied by X-ray analysis.     

Theoretical conformational analysis for neurotransmitters in the gas phase and in aqueous solution. Norepinephrine

The natural neurotransmitter (R)-norepinephrine takes the monocationic form in 93% abundance at the physiological tissue pH of 7.4. Ab initio and DFT/B3LYP calculations were performed for 12 protonated conformers of (R)-norepinephrine in the gas phase with geometry optimizations up to the MP2/6-311++G level, and with single-point calculations up to the QCISD(T) level at the HF/6-31G-optimized geometries. Four monohydrates were studied at the MP2/6-31G//HF/6-31G level. In the gas phase, the G1 conformer is the most stable with phenyl.NH(3)(+) gauche and HO(alc).NH(3)(+) gauche arrangements. A strained intramolecular hydrogen bond was found for conformers (G1 and T) with close NH(3)(+) and OH groups. Upon rotation of the NH(3)(+) group as a whole unit about the C(beta)-C(alpha) axis, a 3-fold potential was calculated with free energies for barriers of 3-12 kcal/mol at the HF/6-31G level. Only small deviations were found in MP2/6-311++G single-point calculations. A 2-fold potential was calculated for the phenyl rotation with free energies of 11-13 kcal/mol for the barriers at T = 310 K and p = 1 atm. A molecular mechanics docking study of (R)-norepinephrine in a model binding pocket of the beta-adrenergic receptor shows that the ligand takes a conformation close to the T(3) arrangement. The effect of aqueous solvation was considered by the free energy perturbation method implemented in Monte Carlo simulations. There are 4-5 strongly bound water molecules in hydrogen bonds to the conformers. Although hydration stabilizes mostly the G2 form with gauche phenyl.NH(3)(+) arrangement and a water-exposed NH(3)(+) group, the conformer population becomes T > G1 > G2, in agreement with the PMR spectroscopy measurements by Solmajer et al. (Z. Naturforsch. 1983, 38c, 758). Solvent effects reduce the free energies for barriers to 3-6 and 9-12 kcal/mol for rotations about the C(beta)-C(alpha) and the C(1)(ring)-C(beta) axes, respectively.     

Glycomimetic ligands for the human asialoglycoprotein receptor

The asialoglycoprotein receptor (ASGPR) is a high-capacity galactose-binding receptor expressed on hepatocytes that binds its native substrates with low affinity. More potent ligands are of interest for hepatic delivery of therapeutic agents. We report several classes of galactosyl analogues with varied substitution at the anomeric, C2-, C5-, and C6-positions. Significant increases in binding affinity were noted for several trifluoromethylacetamide derivatives without covalent attachment to the protein. A variety of new ligands were obtained with affinity for ASGPR as good as or better than that of the parent N-acetylgalactosamine, showing that modification on either side of the key C3,C4-diol moiety is well tolerated, consistent with previous models of a shallow binding pocket. The galactosyl pyranose motif therefore offers many opportunities for the attachment of other functional units or payloads while retaining low-micromolar or better affinity for the ASGPR.     

Excess electrons bound to small ammonia clusters

Dipole-bound anions of small water clusters (H2O) N- (N >or= 2) are well-known from experiment and theory. In contrast, the smallest ammonia cluster anion detected so far is the 13-mer (NH3)13-. Here dipole-bound states of small ammonia clusters (NH3)N- (N = 2, 3, 4) are investigated using coupled-cluster ab initio methods. The trimer is found to be the smallest ammonia cluster able to form a dipole bound state, and its vertical detachment energy is predicted to be 27 meV, somewhat smaller than that of the water dimer. For the ammonia tetramer dipole-bound states with triple-acceptor monmers are identified akin to the well-studied double-acceptor binding motif of water cluster anions. Moreover, a (NH3)6-)hexamer that has been considered as a model for a cavity-bound state is examined. Ab initio results for this system challenge the notion that an electron localized in an ammonia cavity can be thought of as a delocalized radical anion.     

Recherches sur la formation et la transformation des esters LXXII. Aryl(ou aralcoyl ou alcoyl)amino-2-tétrahydro-m-thiazines ou aryl(ou aralcoyl ou alcoyl)amino-2-dihydro-Δ2-m-thiazines et dérivés

3-Aminopropanol reacts with aryl(or aralkyl or alkyl)isothiocyanates R? N?C?S to yield the corresponding thio-ureas R? NH? CS? NH? (CH₂)₃OH which, refluxed with hydrochloric acid, are cyclized by elimination of water. The cyclization products are identical with the hydrothiazines resulting by elimination of sulfate or phosphate from the sulfuric or phosphoric monoesters of these thio-ureas. The resulting hydrothiazines are either 2-(R-imino)-tetrahydro-m-thiazines (I) or 2-(R-amino)-dihydro-Δ²-m-thiazines (II). Their structure has been established by comparison of their spectra with those of model compounds in one of which the C?N double bond is certainly endocyclic (2-methyl-dihydro-Δ²-m-thiazine), the other presenting an exocyclic C?N double bond (3-methyl-2-phenylimino-tetrahydro-m-thiazine). When R is an aryl group, the C?N double bond is exocyclic (structure I with >C?N? Ar), and one may presume that this structure is stabilized by resonance. When R is an aralkyl or an alkyl group, the C?N double bond is endocyclic (structure II). The nmr spectra were taken with three types of solvent: CDCl₃ or CCl₄; (CD₃)₂SO; CF₃COOH. In CF₃COOH solution the benzylic protons of the hydrothiazine with R = pF? C₆H₄CH₂? couple with NH (J=5,5cps) which confirms the endocyclic position of the C?N double bond in this case.     

Hydrogen bonding interactions of pyridine*+ with water: stepwise solvation of distonic cations

The solvation energies of the pyridine*+ radical cation by 1-4 H2O molecules were determined by equilibrium measurements in a drift cell. The binding energies of the pyridine*+(H2O)n clusters are similar to the binding energies of protonated pyridine-water clusters, (C5H5NH+)(H2O)n, which involve NH+..OH2 bonds and different from those of the solvated benzene radical cation-water clusters, C6H6*+(H2O)n, which involve CHdelta+..OH2 bonds. These relations indicate that the observed pyridine*+ ions have the distonic *C5H4NH+ structures that can form NH+..OH2 bonds. The observed thermochemistry and ab initio calculations show that these bonds are not affected significantly by an unpaired electron at another site of the ion. Similar observations also identify the 2-fluoropyridine*+ distonic ion. The distonic structure is also consistent with the reactivity of pyridine*+ in H atom transfer, intra-cluster proton transfer and deprotonation reactions. The results present the first measured stepwise solvation energies of distonic ions, and demonstrate that cluster thermochemistry can identify distonic structures.     

Conversion of a Re(IV) tetrahedral cluster to a Re(III) octahedral cluster: synthesis of [(CH3)C(NH2)(2)](4)[Re(6)Se(8)(CN)(6)] by a solvothermal route

The compound [(CH(3))C(NH(2))(2)](4)[Re(6)Se(8)(CN)(6)] has been synthesized by the reaction at 200 degrees C for 3 days of Re(4)Te(4)(TeCl(2))(4)Cl(8), KSeCN, and NH(4)Cl in superheated acetonitrile. This compound crystallizes in the space group C2/c of the monoclinic system with four formula units in a cell of dimensions a = 20.3113(14) A, b = 10.1332(7) A, c = 19.9981(14) A, beta = 106.754(1) degrees, V = 3941.3(5) A(3) (T = 153 K). The [Re(6)Se(8)(CN)(6)](4-) anion comprises an Re(6) octahedron face capped by mu(3)-Se atoms, with each Re atom liganded by a CN group. The anions and cations are connected by an extensive network of hydrogen bonds. The conversion of a Re(IV) tetrahedral cluster to a Re(III) octahedral cluster appears to be unprecedented.     

Adenosine A2A receptor agonists: CoMFA-based selection of the most predictive conformation

A step-wise comparative molecular field analysis (CoMFA)-based procedure was applied to a series of 51 2-oxyadenosines in order to select the most predictive conformation for binding to A2A adenosine receptor (AR). The highest correlation and predictive power were found for conformers with side chain at 2nd position oriented in the direction opposite to the exocyclic amino group on the adenine ring (torsion N1C2OR = 120 degrees) and fully extended. The interaction of ligand and receptor is under steric and electrostatic control. The steric contribution is of a greater importance for the predictivity than the electrostatic one. Hydrophobicity of the compounds investigated does not affect significantly either the affinity to A2A AR, nor the predictivity of the models.     

Electronic Structure of Six-Membered N-Heterocyclic Carbenes and Its Heavier Analogues: Reactivity of the Lone Pair versus the Exocyclic Double Bond

Electronic structure of the six-membered N-heterocyclic carbene, silylene, germylene, and stannylene having an exocyclic double bond at the C3 carbon atom as well as the relative reactivity of the lone-pair on the divalent group 14 element and the exocyclic double bond have been studied at the BP86 level of theory with a TZVPP basis set. The geometrical parameters, NICS values, and NBO population analysis indicate that these molecules can be best described as the localized structure 1X(a), where a trans-butadiene (C1-C2-C3-C4) unit is connected with diaminocarbene (N1-X-N2) via N-atoms having a little contribution from the delocalized structure 1X(b). The proton affinity at X is higher than at C4 for 1C, and a reverse trend is observed for the heavier analogues. Hence, the lone pair on a heavier divalent Group 14 element is less reactive than the exocyclic double bond. This is consistent with the argument that, even though the parent six-membered carbene and its heavier analogues are nonaromatic in nature, the controlled and targeted protonation can lead to either the aromatic system 3X having a lone pair on X or the nonaromatic system 2X with readily polarizable C3-C4 π-bond. The energetics for the reaction with BH(3) and W(CO)(6) further suggest that both the lone pair of Group 14 element and the exocyclic double bond can act as Lewis basic positions, although the reaction at one of the Lewis basic positions in 1X does not considerably influence the reactivity at the other. The protonation and adduct formation with BH(3) and W(CO)(5) at X lead to nonaromatic systems whereas similar reactions at C4 lead to aromatic systems due to π-bond polarization at C3-C4. The degree of polarization of the C3-C4 π-bond is maximum in the protonated adduct and reduces in the complexes formed with BH(3) and W(CO)(5).     

DFT investigation of NH3 physisorption on CuSO4 impregnated SiO2

In this quantum chemical investigation, NH(3) physisorption onto a model of copper sulfate impregnated silica is compared with pure silica and copper sulfate adsorbents. The physisorption process is modeled as direct binding of the NH(3) molecule to the adsorption site of the dry adsorbents and as displacement of a H(2)O molecule by NH(3) in the hydrated complexes. The surface of silica is represented by a hydroxyl group attached to a silsesquioxane cage, H(7)Si(8)O(12)(OH) and silica impregnated with CuSO(4) by the most stable configuration of the cluster containing a CuSO(4) ion pair placed adjacent to the silica cage. H(2)O is systematically added to the dehydrated adsorbents to investigate the role of water in NH(3) adsorption. Modeling hydrated environments of each type of adsorbent is focused on H(2)O molecules that directly coordinate with the active sites. The results indicate that the binding energy of adsorbing NH(3) onto the mixed adsorbent is greater than in pure silica. This enhanced binding in the mixed adsorbent is consistent with improved Br?nsted acidity of the silanol in the presence of CuSO(4).     

Adenosine A 2A receptor agonists: CoMFA-based selection of the most predictive conformation

A step-wise comparative molecular field analysis (CoMFA)-based procedure was applied to a series of 51 2-oxyadenosines in order to select the most predictive conformation for binding to A 2A adenosine receptor (AR). The highest correlation and predictive power were found for conformers with side chain at 2nd position oriented in the direction opposite to the exocyclic amino group on the adenine ring (torsion N1C2OR=120 ) and fully extended. The interaction of ligand and receptor is under steric and electrostatic control. The steric contribution is of a greater importance for the predictivity than the electrostatic one. Hydrophobicity of the compounds investigated does not affect significantly either the affinity to A 2A AR, nor the predictivity of the models.     

C4H5N-(NH3)n氢键团簇的多光子电力与从头计算

在３５５和５３２ｎｍ激光波长下用ＴＯＦ质谱仪研究了Ｃ４Ｈ５Ｎ－（ＮＨ３）ｎ系列氢键团簇体系的多光子电离,实验发现,两波长下除了得到一系列团簇离子Ｃ４Ｈ５Ｎ－（ＮＨ３）ｎ＾＋外,还观测到一系列质子化产物Ｃ４Ｈ５Ｎ－（ＮＨ３）ｎ－Ｈ＾＋,这些质子化产物来自于光电离过程中团簇内部的质子转移反应;Ｃ４Ｈ５Ｎ－（ＮＨ３）ｎ＾＋系列离子出现反常强度变化,即Ｃ４Ｈ５Ｎ－（ＮＨ３）２＾＋离子强度较Ｃ４Ｈ５Ｎ－（Ｎ     

Facilitated phosphatidylserine (PS) flip-flop and thrombin activation using a synthetic PS scramblase

A cationic steroid with a hydrogen-bonding pocket that has an affinity for anionic phospholipid headgroups was synthesized and shown to strongly promote the translocation or flip-flop of a fluorescent, C(6)NBD-labeled phosphatidylserine probe (C(6)NBD-PS) across vesicle membranes. In addition, the synthetic PS scramblase increases the levels of endogenous PS on the surface of erythrocytes as monitored by flow cytometry analysis of annexin V-FITC binding. The PS scrambling effect is enhanced when the cells are pretreated with N-ethylmaleimide (NEM), an inhibitor of the endogenous aminophospholipid flippase. The combination of NEM and synthetic PS scramblase enhances the ability of erythrocytes to promote the conversion of prothrombin to thrombin by a factor of 4. An analogous cationic steroid with a smaller binding pocket has no measurable PS translocation activity, a result that is attributed to its inability to sufficiently diminish the hydrophilicity of the multiply charged PS headgroup.     

Screening for galectin-3 inhibitors from synthetic lacto-N-biose libraries using microscale affinity chromatography coupled to mass spectrometry

The synthesis and screening of two beta-D-Galp-(1-3)-beta-d-GlcpN (lacto-N-biose) disaccharide libraries are reported. Solution-phase synthetic modifications at the HO-2' and NH positions were performed in an effort to enhance the affinity toward galectin-3, a galactose-binding protein involved in tumor metastasis, apoptosis, and inflammation. The libraries were screened for galectin-3 binding by microscale frontal affinity chromatography coupled to mass spectrometry (FAC/MS) allowing for rapid ranking of the different inhibitors and the determination of the galectin-3 binding Kd's. Compounds bearing a hydrophobic substituent on the NH group showed the highest affinity for the lectin. The N-naphthoyl derivative (Kd = 10.6 microM) was the best inhibitor with a 7 times increased affinity as compared to the N-acetyl parent compound (Kd = 73.3 microM).     

A disaccharide rearrangement catalyzed by molybdate anion in aqueous solution

Reaction of a galactosylated 2-C-(hydroxymethyl)-tetrofuranose with paramolybdate ion-exchange resin in aqueous solution at 67 degrees C gave an equililibrium mixture containing the reactant aldofuranose (42%) and a 2-ketopentofuranose galactosylated at O1 (58%). Observation of this stereospecific rearrangement supports prior arguments that substituents at C2 of the branched-chain aldofuranose reactant are located in a sterically accessible pocket of the putative dimolybdate-saccharide reactive complex during epimerization. This rearrangement provides a new and convenient route to 2-ketosugars glycosylated at the exocyclic C1 position.