Synthesis,Biological Evaluation,and Crystallographic Studies of Extended Guanine‐Based (lin‐Benzoguanine) Inhibitors for tRNA‐Guanine Transglycosylase (TGT)

This paper describes the rational design, synthesis, and biological evaluation of a new generation of inhibitors of the bacterial enzyme tRNA‐guanine transglycosylase (TGT), which has been identified as a new target in the fight against bacillary dysentery (Shigellosis). The enzyme catalyzes the exchange of guanine in the anticodon wobble position of tRNA by the modified base preQ₁, a guanine derivative, according to a ping‐pong mechanism involving a covalent TGT‐tRNA intermediate (Fig. 2). Based on computer modeling (Fig. 3), lin‐benzoguanine (6‐aminoimidazol[4,5‐g]quinazolin‐8(7H)‐one ( 2 )) was selected as an extended central scaffold, to form up to seven in‐plane intermolecular H‐bonds with the protein while sandwiching between Tyr106 and Met260. Versatile synthetic protocols were developed for the synthesis of 2 , and derivatives with phenyl, benzyl, and 2‐phenylethyl side chains (i.e., 16, 17a , and 12a, 12b, 13, 17 , resp.) to reach into the lipophilic pocket lined by Val282, Val45, and Leu68 (Schemes 1–3). To account for the limited solubility of the new ligands and in consequence of a recently developed detailed understanding of the mechanism of TGT catalysis (Fig. 2), the enzyme kinetic assay was completely redesigned, providing competitive (K_ic) and uncompetitive (K_iu) inhibition constants with respect to tRNA binding by TGT. The modifications of the various parameters in the new assay are described in detail. Binding affinities of the new inhibitors were found to be in the single‐digit micromolar range (K_ic values, Fig. 8). Decoration of the lin‐benzoguanine scaffold with lipophilic residues only gave a modest improvement in biological activity which was explained on structural grounds with the help of four crystal structures (Fig. 10) obtained by soaking the protein with inhibitors 2 and 12a – 12c . Both biochemical and biostructural analyses reported in this paper provide a fertile basis for the development of more potent future generations of TGT inhibitors.     

An Immucillin‐Based Transition‐State‐Analogous Inhibitor of tRNA–Guanine Transglycosylase (TGT)

Shigellosis is one of the most severe diarrheal diseases worldwide without any efficient treatment so far. The enzyme tRNA–guanine transglycosylase (TGT) has been identified as a promising target for small‐molecule drug design. Herein, we report a transition‐state analogue, a small, immucillin‐derived inhibitor, as a new lead structure with a novel mode of action. The complex inhibitor synthesis was accomplished in 18 steps with an overall yield of 3 %. A co‐crystal structure of the inhibitor bound to Z. mobilis TGT confirmed the predicted conformation of the immucillin derivative in the enzyme active site.     

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.     

DNA脱碱基位点存在下非氢键配体的增强结合研究

众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基（AP）位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素（proflavine,PF）为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。     

Site‐Specific Investigation of the Steady‐State Kinetics and Dynamics of the Multistep Binding of Bile Acid Molecules to a Lipid Carrier Protein

The investigation of multi‐site ligand–protein binding and multi‐step mechanisms is highly demanding. In this work, advanced NMR methodologies such as 2D ¹H–¹⁵N line‐shape analysis, which allows a reliable investigation of ligand binding occurring on micro‐ to millisecond timescales, have been extended to model a two‐step binding mechanism. The molecular recognition and complex uptake mechanism of two bile salt molecules by lipid carriers is an interesting example that shows that protein dynamics has the potential to modulate the macromolecule–ligand encounter. Kinetic analysis supports a conformational selection model as the initial recognition process in which the dynamics observed in the apo form is essential for ligand uptake, leading to conformations with improved access to the binding cavity. Subsequent multi‐step events could be modelled, for several residues, with a two‐step binding mechanism. The protein in the ligand‐bound state still exhibits a conformational rearrangement that occurs on a very slow timescale, as observed for other proteins of the family. A global mechanism suggesting how bile acids access the macromolecular cavity is thus proposed.     

Delineating Binding Modes of Gal/GalNAc and Structural Elements of the Molecular Recognition of Tumor‐Associated Mucin Glycopeptides by the Human Macrophage Galactose‐Type Lectin

The human macrophage galactose‐type lectin (MGL) is a key physiological receptor for the carcinoma‐associated Tn antigen (GalNAc‐α‐1‐O‐Ser/Thr) in mucins. NMR and modeling‐based data on the molecular recognition features of synthetic Tn‐bearing glycopeptides by MGL are presented. Cognate epitopes on the sugar and matching key amino acids involved in the interaction were identified by saturation transfer difference (STD) NMR spectroscopy. Only the amino acids close to the glycosylation site in the peptides are involved in lectin contact. Moreover, control experiments with non‐glycosylated MUC1 peptides unequivocally showed that the sugar residue is essential for MGL binding, as is Ca²⁺. NMR data were complemented with molecular dynamics simulations and Corcema‐ST to establish a 3D view on the molecular recognition process between Gal, GalNAc, and the Tn‐presenting glycopeptides and MGL. Gal and GalNAc have a dual binding mode with opposite trend of the main interaction pattern and the differences in affinity can be explained by additional hydrogen bonds and CH–π contacts involving exclusively the NHAc moiety.     

Identification and Characterization of a Single High‐Affinity Fatty Acid Binding Site in Human Serum Albumin

A single high‐affinity fatty acid binding site in the important human transport protein serum albumin (HSA) is identified and characterized using an NBD (7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐C₁₂ fatty acid. This ligand exhibits a 1:1 binding stoichiometry in its HSA complex with high site‐specificity. The complex dissociation constant is determined by titration experiments as well as radioactive equilibrium dialysis. Competition experiments with the known HSA‐binding drugs warfarin and ibuprofen confirm the new binding site to be different from Sudlow‐sites I and II. These binding studies are extended to other albumin binders and fatty acid derivatives. Furthermore an X‐ray crystal structure allows locating the binding site in HSA subdomain IIA. The knowledge about this novel HSA site will be important for drug depot development and for understanding drug‐protein interaction, which are important prerequisites for modulation of drug pharmacokinetics.     

Tuning the Redox Properties of a Nonheme Iron(III)–Peroxo Complex Binding Redox‐Inactive Zinc Ions by Water Molecules

Redox‐inactive metal ions play important roles in tuning chemical properties of metal–oxygen intermediates. Herein we report the effect of water molecules on the redox properties of a nonheme iron(III)–peroxo complex binding redox‐inactive metal ions. The coordination of two water molecules to a Zn²⁺ ion in (TMC)Fe^III‐(O₂)‐Zn(CF₃SO₃)₂ ( 1 ‐Zn²⁺) decreases the Lewis acidity of the Zn²⁺ ion, resulting in the decrease of the one‐electron oxidation and reduction potentials of 1 ‐Zn²⁺. This further changes the reactivities of 1 ‐Zn²⁺ in oxidation and reduction reactions; no reaction occurred upon addition of an oxidant (e.g., cerium(IV) ammonium nitrate (CAN)) to 1 ‐Zn²⁺, whereas 1 ‐Zn²⁺ coordinating two water molecules, (TMC)Fe^III‐(O₂)‐Zn(CF₃SO₃)₂‐(OH₂)₂ [ 1 ‐Zn²⁺‐(OH₂)₂], releases the O₂ unit in the oxidation reaction. In the reduction reactions, 1 ‐Zn²⁺ was converted to its corresponding iron(IV)–oxo species upon addition of a reductant (e.g., a ferrocene derivative), whereas such a reaction occurred at a much slower rate in the case of 1 ‐Zn²⁺‐(OH₂)₂. The present results provide the first biomimetic example showing that water molecules at the active sites of metalloenzymes may participate in tuning the redox properties of metal–oxygen intermediates.     

Molecular recognition at the active site of catechol-O-methyltransferase (COMT): adenine replacements in bisubstrate inhibitors

L-Dopa, the standard therapeutic for Parkinson's disease, is inactivated by the enzyme catechol-O-methyltransferase (COMT). COMT catalyzes the transfer of an activated methyl group from S-adenosylmethionine (SAM) to its catechol substrates, such as L-dopa, in the presence of magnesium ions. The molecular recognition properties of the SAM-binding site of COMT have been investigated only sparsely. Here, we explore this site by structural alterations of the adenine moiety of bisubstrate inhibitors. The molecular recognition of adenine is of special interest due to the great abundance and importance of this nucleobase in biological systems. Novel bisubstrate inhibitors with adenine replacements were developed by structure-based design and synthesized using a nucleosidation protocol introduced by Vorbrüggen and co-workers. Key interactions of the adenine moiety with COMT were measured with a radiochemical assay. Several bisubstrate inhibitors, most notably the adenine replacements thiopyridine, purine, N-methyladenine, and 6-methylpurine, displayed nanomolar IC(50) values (median inhibitory concentration) for COMT down to 6 nM. A series of six cocrystal structures of the bisubstrate inhibitors in ternary complexes with COMT and Mg(2+) confirm our predicted binding mode of the adenine replacements. The cocrystal structure of an inhibitor bearing no nucleobase can be regarded as an intermediate along the reaction coordinate of bisubstrate inhibitor binding to COMT. Our studies show that solvation varies with the type of adenine replacement, whereas among the adenine derivatives, the nitrogen atom at position 1 is essential for high affinity, while the exocyclic amino group is most efficiently substituted by a methyl group.     

Synthesis,Crystal Structure,Antioxidation and DNA‐Binding Studies of a Zinc(II) Complex with the Bis(N‐allylbenzimidazol‐2‐ylmethyl)aniline

A complex of zinc(II) picrate (pic) with bis(N‐allylbenzimidazol‐2‐ylmethyl)aniline (abba), with composition [Zn(abba)₂](pic)₂, was synthesized and characterized by elemental analysis, electrical conductivity, IR and UV/Vis spectral measurements. The crystal structure of the zinc(II) complex has been determined by single‐crystal X‐ray diffraction. The Zn(II) is bonded to two abba ligands through four benzimidazole nitrogen, resulting in a distorted tetrahedron geometry. The DNA‐binding properties of the ligand and the zinc(II) complex were investigated by electronic absorption, fluorescence spectra and viscosity measurements. The experimental results suggest that the zinc(II) complex binds to DNA in an intercalation mode. In addition, the ligand abba and Zn(II) complex have scavenging effects for hydroxyl radicals and the complex shows stronger scavenging effects for hydroxyl radicals than the ligand.     

Binding Behaviors for Different Types of DNA G‐Quadruplexes: Enantiomers of [Ru(bpy)2(L)]2+ (L=dppz,dppz‐idzo)

Polymorphic DNA G‐quadruplex recognition has attracted great interest in recent years. The strong binding affinity and potential enantioselectivity of chiral [Ru(bpy)₂(L)]²⁺ (L=dipyrido[3,2‐a:2′,3′‐c]phenazine, dppz‐10,11‐imidazolone; bpy=2,2′‐bipyridine) prompted this investigation as to whether the two enantiomers, Δ and Λ, can show different effects on diverse structures with a range of parallel, antiparallel and mixed parallel/antiparallel G‐quadruplexes. These studies provide a striking example of chiral‐selective recognition of DNA G‐quadruplexes. As for antiparallel (tel‐Na⁺) basket G‐quadruplex, the Λ enantiomers bind stronger than the Δ enantiomers. Moreover, the behavior reported here for both enantiomers stands in sharp contrast to B‐DNA binding. The chiral selectivity toward mixed parallel/antiparallel (tel‐K⁺) G‐quadruplex of both compounds is weak. Different loop arrangements can change chiral complex selectivity for both antiparallel and mixed parallel/antiparallel G‐quadruplex. Whereas both Δ and Λ isomers bind to parallel G‐quadruplexes with comparable affinity, no appreciable stereoselective G‐quadruplex binding of the isomers was observed. In addition, different binding stoichiometries and binding modes for Δ and Λ enantiomers were confirmed. The results presented here indicate that chiral selective G‐quadruplex binding is not only related to G‐quadruplex topology, but also to the sequence and the loop constitution.     

Delineation of G‐Quadruplex Alkylation Sites Mediated by 3,6‐Bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole‐Aniline Mustard Conjugates

A new G‐quadruplex (G‐4)‐directing alkylating agent BMVC‐C3M was designed and synthesized to integrate 3,6‐bis(1‐methyl‐4‐vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various telomeric G‐4 structures (hybrid‐2 type and antiparallel) and an oncogene promoter, c‐MYC (parallel), were constructed to react with BMVC‐C3M, yielding 35 % alkylation yield toward G‐4 DNA over other DNA categories (<6 %) and high specificity under competition conditions. Analysis of the intact alkylation adducts by electrospray ionization mass spectroscopy (ESI‐MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross‐linking sites were determined and found to be dependent on G‐4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC‐C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c‐MYC), respectively, as monoalkylated adducts and formed A15–C3M–A21 (H26), G12–C3M–G4 (H24), and G2–C3M–G4/G17 (c‐MYC), respectively, as cross‐linked dialkylated adducts. Collectively, the stability and site‐selective cross‐linking capacity of BMVC‐C3M provides a credible tool for the structural and functional characterization of G‐4 DNAs in biological systems.     

Tuning the Guest‐Binding Ability of a Helically Folded Capsule by In Situ Modification of the Aromatic Oligoamide Backbone

Starting from a previously described aromatic oligoamide helically folded capsule that binds tartaric acid with high affinity and diastereoselectivity, we demonstrate the feasibility of the direct in situ modification of the helix backbone, which results in a conformational change that reduces its affinity for guests by two orders of magnitude. Specifically, ring contraction of the central pyridazine unit into a pyrrole in the full helical sequence was investigated by using electrochemical and chemical processes. The sequence containing the pyrrole was synthesized independently in a convergent manner to ascertain its structure. The conformation of the pyrrolic folded capsule was elucidated in the solid state by X‐ray crystallography and in solution by using ¹H and ¹³C NMR spectroscopy. Solution studies revealed an unanticipated solvent‐dependent equilibrium between the anti–anti and syn–syn conformations of the pyrrole ring with respect to its two adjacent pyridine units. Titrations of the pyrrole‐containing sequence monitored by ¹H NMR spectroscopy confirmed the expected drop in affinity for tartaric acid and malic acid that arises from the conformation change in the backbone that follows the replacement of the pyridazine by a pyrrole. The reduction of the pyridazine to a pyrrole was characterized by cyclic voltammetry both on the entire sequence and on a shorter precursor. The lower cathodic potential of the precursor made its preparative‐scale electroreduction possible. Direct in situ modification of the pyridazine within the entire capsule sequence was achieved chemically by using zinc in acetic acid.     

Adsorption of Tri‐n‐Butyl Phosphate (TBP) from NaClO4 Aqueous Solution on Hg Electrode

The adsorption of TBP on Hg electrode from aqueous NaClO₄ media has been studied using differential capacity and chronocoulometric measurements. TBP adsorbs in two steps, the first corresponding to a flat and second to a perpendicular orientation. Both orientations were observed in the entire investigated potential range. The maximal measured surface concentration (Γ_max) of TBP reaches a value of 1.55×10^?10 mol cm^?2, which corresponds to the theoretical value for densely packed molecules in perpendicular orientation. Γ value of the first isotherm step plateau is about 7 to 15% smaller than Γ_max and decreases strongly at potentials more negative than ?0.9 V due to the repelling of negative charges between the electrode and the phosphate group. The standard Gibbs energy of adsorption was found to reach the value of ?34 kJ mol^?1.     

4, 5-二氮芴-9-酮Cu(II)、Co(II)多核配合物的合成，晶体结构及DNA结合作用初探

合成了两个4, 5-二氮芴-9-酮Cu(II)、Co(II)的多核配合物[Cu2(CH3COO)4(H2O) 2]·2dafo 1 和 [(μ2-O)2-Co3(dafo)6] (ClO3)2·H2O 2 (dafo=4,5-diazafluoren-9-one) 并且对它们进行了元素分析,红外以及紫外光谱的表征,同时测定了配合物的晶体结构。用紫外光谱,发射光谱和循环伏安三种方法初步研究了配合物1和2与DNA的结合作用,结果表明,配合物1和2与DNA的结合为以插入作用为主 。