Molecular recognition of duplex DNA by proteins plays a central role in biology. Although there are no clear and simple rules or codes to describe sequence-specific recognition, hydrogen bonds with the nucleic bases as well as with the phosphodiester backbone are of critical importance.[1] Efforts to mimic such recognition with smaller synthetic biopolymers have been described. [2] However, in all these cases, specific contacts with the DNA duplex are only provided by hydrogen bonds with the bases, and potential interactions with the DNA backbone are left unexploited. Here we report that 2'-aminoethoxy-modified oligonucleotides can interact simultaneously with the bases and the phosphodiester backbone at each base-pair step of the DNA target, which provides for a dramatic increase in the binding affinity as well as in the association rate constant.The sequence-specific recognition of duplex DNA by pyrimidine oligonucleotides involves the formation of triplehelical structures, which are stabilized by Hoogsteen hydrogen bonds between the bases on the DNA target and the pyrimidine third strand.[3] Examination of a molecular model of a triple helix with a RNA third strand indicates that the 2'-hydroxyl groups of the RNA and the phosphate groups of the DNA second strand are in close proximity.[4] Thus, it appeared conceivable that the attachment of a short amino alkyl group at the 2' position of the ribose of the third strand would allow the protonated amino group to form a specific intermolecular contact with a proximal phosphate group of the DNA duplex. Such charge ± charge interactions should strongly enhance the affinity of the modified oligonucleotide for double-strand DNA.To test this hypothesis, we first examined the properties of a 2'-aminoethoxy-modified oligonucleotide with regard to triplex formation. The synthesis of the 2'-aminoethyl-modified monomeric tymidine and C5-methylcytidine building blocks is summarized in Scheme 1. Alkylation of protected ribothymidine 1[5] with methyl bromoacetate followed by reduction of the ester group and tosylation gave the tosylate 2. Hydrogenolytic removal of the BOM group and subsequent replacement of the tosyloxy group by an azide substituent yielded the azidoethyl derivative 3, which was further transformed into the desired protected 2'-aminoethoxy-thymidine Scheme 1. Synthesis of protected 2'-aminoethoxy phosphoramidites thymidine (5) and C5-methylcytidine (8): a) BrCH 2 CO 2 Me (5 equiv), NaH (2.2 equiv), DMF, 1.5 h, À 5 8C, 98 %; b) LiBH 4 (4 equiv), MeOH/THF (2/ 8), 1.5 h, 5 8C, 84 %; c) TsCl (1.5 equiv), NEt 3 (1.6 equiv), DMAP (10 wt %), CH 2 Cl 2 , 8 h, 22 8C, 89 %; d) H 2 , Pd/C (20 wt %), HCl (0.02 equiv), THF/ MeOH (1/1), 7 h, 22 8C, 88 %; e) NaN 3 (3 equiv), DMF, 3 h, 65 8C, 92 %; f) SnCl 2 (3.5 equiv), MeOH, 1 d, 22 8C, 70 %; g) (CF 3 CO) 2 O (1.2 equiv), pyridine, 2 h, 22 8C, 57 %; h) TBAF (2.1 equiv), THF, 15 min, 22 8C, 100 %; i) DMTrCl (1.2 equiv), pyridine, 16 h, 22 8C, 85 %; j) (iPr 2 N) 2 -POCH 2 CH 2 CN (2.2 equiv), diisopropylammonium tetrazolide (...
This paper reports a comparison of calculated molecular properties and of 2D fragment bit-strings when used for the selection of structurally diverse subsets of a file of 44295 compounds. MaxMin dissimilarity-based selection and k-means clusterbased selection are used to select subsets containing between 1% and 20% of the file. Investigation of the numbers of bioactive molecules in the selected subsets suggest: that the MaxMin subsets are noticeably superior to the k-means subsets; that the property-based descriptors are marginally superior to the fragment-based descriptors; and that both approaches are noticeably superior to random selection.
The conformational features of sucrose in the combining site of lentil lectin have been characterized through elucidation of a crystalline complex at 1.9-Å resolution, transferred nuclear Overhauser effect experiments performed at 600 Mhz, and molecular modeling. In the crystal, the lentil lectin dimer binds one sucrose molecule per monomer. The locations of 229 water molecules have been identified. NMR experiments have provided 11 transferred NOEs. In parallel, the docking study and conformational analysis of sucrose in the combining site of lentil lectin indicate that three different conformations can be accommodated. Of these, the orientation with lowest energy is identical with the one observed in the crystalline complex and provides good agreement with the observed transferred NOEs. These structural investigations indicate that the bound sucrose has a unique conformation for the glycosidic linkage, close to the one observed in crystalline sucrose, whereas the fructofuranose ring remains relatively flexible and does not exhibit any strong interaction with the protein. Major differences in the hydrogen bonding network of sucrose are found. None of the two inter-residue hydrogen bonds in crystalline sucrose are conserved in the complex with the lectin. Instead, a water molecule bridges hydroxyl groups O2-g and O3-f of sucrose.
The three-dimensional structure of Dolichos biflorus seed lectin has been constructed using five legume lectins for which high resolution crystal structures were available. The validity of the resulting model has been thoroughly investigated. Final structure optimization was conducted for the lectin complexed with alpha GalNAc, providing thereby the first three-dimensional structure of lectin/GalNAc complex. The role of the N-acetyl group was clearly evidenced by the occurrence of a strong hydrogen bond between the protein and the carbonyl oxygen of the carbohydrate and by hydrophobic interaction between the methyl group and aromatic amino acids. Since the lectin specificity is maximum for the Forssman disaccharide alpha GalNAc(1-3) beta GalNAc-O-Me and the blood group A trisaccharide alpha GalNAc(1-3)[alpha Fuc(1-2)] beta Gal-O-Me, the complexes with these oligosaccharides have been also modelled.
The present study is concerned with the elucidation of the conformation of the blood group A trisaccharide (u-~-GalNAc(l-+3)[a-~-Fuc(l*2)]~-~-Gal-O-R) in the combining site of Dolichos bifZorus seed lectin by use of 400-MHz and 600-MHz NMR spectroscopy. D. b$!oru.s lectin displays a unique specificity for CalNAc residues. It occurs in solution as a tetranieric assembly having a molecular mass of 1 I0 kDa, with two carbohydrate-binding sites per molecule. First, NOE build-up curves were obtained for the free blood group A trisaccharide from one-dimensional transient NOE experiments. Simulated NOE build-up curves were constructed from an ensemble of low-energy conformers derived from previous investigations. The comparison of theoretical and experimental data indicates that an equilibrium between two families of low-energy conformers most likely reflects the solution behavior of the trisaccharide in solution. Two-dimensional transferred NOE and rotating-frame enhancements (ROE) were subsequently measured for the trisaccharide complexed with the D. biJZorus seed lectin. In addition to the NOEs observed for the free trisaccharide, the transferred NOESY spectrum showed several new NOEs that were identified as spin diffusion using a rotating-frame NOESY (ROESY) experiment. Experimental interglycosidic transferred nuclear Overhauser effect (TRNOE) build-up curves were compared to theoretical curves calculated for both low-energy conformers located in the D. biflorus lectin-binding site. Calculations of theoretical TRNOE were performed using a combination of the full relaxation matrix and the protein-ligand exchange matrix. Comparison between experimental and simulated TRNOE volumes leads to the conclusion that one conformation of blood group A trisaccharide is selected upon binding by D. bi9oru.s lectin.
The X-ray crystal structure of lentil lectin in complex with alpha-D-glucopyranose has been determined by molecular replacement and refined to an R-value of 0.20 at 3.0 A resolution. The glucose interacts with the protein in a manner similar to that found in the mannose complexes of concanavalin A, pea lectin and isolectin I from Lathyrus ochrus. The complex is stabilized by a network of hydrogen bonds involving the carbohydrate oxygens O6, O4, O3 and O5. In addition, the alpha-D-glucopyranose residue makes van der Waals contacts with the protein, involving the phenyl ring of Phe123 beta. The overall structure of lentil lectin, at this resolution, does not differ significantly from the highly refined structures of the uncomplexed lectin. Molecular docking studies were performed with mannose and its 2-O and 3-O-m-nitro-benzyl derivatives to explain their high affinity binding. The interactions of the modelled mannose with lentil lectin agree well with those observed experimentally for the protein-carbohydrate complex. The highly flexible Me-2-O-(m-nitro-benzyl)-alpha-D-mannopyranoside and Me-3-O-(m-nitro-benzyl)-alpha-D-mannopyranoside become conformationally restricted upon binding to lentil lectin. For best orientations of the two substrates in the combining site, the loss of entropy is accompanied by the formation of a strong hydrogen bond between the nitro group and one amino acid, Gly97 beta and Asn125 beta, respectively, along with the establishment of van der Waals interactions between the benzyl group and the aromatic amino acids Tyr100 beta and Trp128 beta.
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