Solution structure by NMR and molecular dynamics of a duplex containing a guanine opposite a N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide lesion

One- and two-dimensional NMR spectroscopy has been used combined with molecular dynamics to determine the fine structure of the DNA duplex 5'-d(AGGAGCCACG).d(CGTGGFTCCT) where F is the N-(2-deoxy-beta-D-erythro-pentofuranosyl)formamide residue which is a ring fragmentation product of thymine. The formamide deoxyribose exists as two isomers with respect to the orientation about the peptide bond. The two isomers (trans and cis) are observed in a ratio 3:2 in solution. For both species, the oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. The formamide residue, whatever the isomer, is intrahelical and can pair with the guanine on the opposite strand with one hydrogen bond. For the cis isomer, the residue adopts a syn orientation and is able to form a second hydrogen bond with the guanine on the 5' side on the same strand. Off-resonance ROESY experiments have been used to investigate the chemical exchange observed at low temperature of the duplex. Conformational exchange has only been found for the oligonucleotide with the formamide residue in the trans conformation.     

Structural study of DNA duplex containing an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide frameshift by NMR and restrained molecular dynamics

The presence of an N-(2-deoxy-beta-D-erythro-pentofuranosyl) formamide (F) residue, a ring fragmentation product of thymine, in a frameshift context in the sequence 5'-d-(AGGACCACG)*d(CGTGGFTCCT) has been studied by 1H and 31P nuclear magnetic resonance (NMR) and molecular dynamics. Two-dimensional NMR studies show that the formamide residue, whether the cis or trans isomer, is rotated out of the helix and that the bases on either side of the formamide residue in the sequence, G14 and T16, are stacked over each other in a way similar to normal B-DNA. The cis and trans isomers were observed in the ratio 3:2 in solution. Information extracted from 31P NMR data reveal a modification of the phosphodiester backbone conformation at the extrahelical site, which is also observed during the molecular dynamics simulations.     

Structure of an O-specific polysaccharide of Proteus mirabilis 03, containing N-(2-hydroxyethyl)-D-alanine

O-Specific polysaccharide, obtained by mild acid degradation of the Proteus mirabilis 03 lipopolysaccharide, was dephosphorylated with 48% HF to give a linear polysaccharide and an amino acid, N-(2-hydroxyethyl)-D-alanine. The structure of the polysaccharide was determined by methylation, the Smith degradation and computer-assisted analysis of the 13C NMR spectra of original and dephosphorylated polymers and oligomers. The structure of the amino acid was elucidated by using 1H and 13C NMR spectroscopy and mass spectrometry (applied to the acetylated methyl ester derivative), optical rotation and CD spectrum data and comparison with the synthetic sample. The repeating unit of P. mirabilis 03 O-specific polysaccharide is shown to have the following structure: (formula; see text)     

Solution conformation of an oligonucleotide containing a urea deoxyribose residue in front of a thymine.

Urea residues are produced by ionizing radiation on thymine residues in DNA. We have studied an oligodeoxynucleotide containing a thymine opposite the urea residue, by one and two dimensional NMR spectroscopy. The urea deoxyribose exists as two isomers with respect to the orientation about the peptide bond. For the trans isomer we find that the thymine and urea site are positioned within the helix and are probably hydrogen bonded. The oligonucleotide adopts a globally B form structure although conformational changes are observed around the mismatch site. A minor species is observed, in which the urea deoxyribose and the opposite base adopt an extrahelical position and this corresponds to the isomer cis for the peptide bond.     

Structure of a novel cofactor containing N-(7-mercaptoheptanoyl)-O-3-phosphothreonine

The cofactor required in the methylcoenzyme M methylreductase reaction was shown to be a large molecule with an Mr of 1149.21 in the free acid form. The cofactor, named MRF for methyl reducing factor, was identified from analyses by fast atom bombardment mass spectrometry and 1H, 13C, and 31P NMR spectroscopy as uridine 5'-[N-(7-mercaptoheptanoyl)-O-3-phosphothreonine-P-yl(2-acetamido- 2-deoxy- beta-mannopyranuronosyl)(acid anhydride)]-(1----4)-O-2-acetamido-2-deoxy- alpha-glucopyranosyl diphosphate. MRF contains N-(7-mercaptoheptanoyl)threonine O-3-phosphate (HS-HTP) [No11, K. M., Rinehart, K. L., Tanner, R. S., & Wolfe, R. S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242] and is linked to C-6 of 2-acetamido-2-deoxymannopyranuronic acid of the UDP-disaccharide through a carboxylic-phosphoric anhydride linkage. It is postulated that this bond is responsible for the instability of the molecule and its hydrolysis during isolation. Analyses of Eadie and Hofstee plots of the methylcoenzyme M methylreductase reaction indicate that MRF has a 6-fold lower Km(app) than HS-HTP and a 50% greater Vmax. This suggests that the UDP-disaccharide moiety may be of importance in the binding of MRF to the enzyme active site.     

Characterization of a Trypanosoma brucei RNA cap (guanine N-7) methyltransferase

The m7GpppN cap structure of eukaryotic mRNA is formed by the sequential action of RNA triphosphatase, guanylyltransferase, and (guanine N-7) methyltransferase. In trypanosomatid protozoa, the m7GpppN is further modified by seven methylation steps within the first four transcribed nucleosides to form the cap 4 structure. The RNA triphosphatase and guanylyltransferase components have been characterized in Trypanosoma brucei. Here we describe the identification and characterization of a T. brucei (guanine N-7) methyltransferase (TbCmt1). Sequence alignment of the 324-amino acid TbCmt1 with the corresponding enzymes from human (Hcm1), fungal (Abd1), and microsporidian (Ecm1) revealed the presence of conserved residues known to be essential for methyltransferase activity. Purified recombinant TbCmt1 catalyzes the transfer of a methyl group from S-adenosylmethionine to the N-7 position of the cap guanine in GpppN-terminated RNA to form the m7GpppN cap. TbCmt1 also methylates GpppG and GpppA but not GTP or dGTP. Mutational analysis of individual residues of TbCmt1 that were predicted-on the basis of the crystal structure of Ecm1--to be located at or near the active site identified six conserved residues in the putative AdoMet- or cap-binding pocket that caused significant reductions in TbCmt1 methyltransferase activity. We also report the identification of a second T. brucei RNA (guanine N-7) cap methyltransferase (named TbCgm1). The 1050-amino acid TbCgm1 consists of a C-terminal (guanine N-7) methyltransferase domain, which is homologous with TbCmt1, and an N-terminal guanylyltransferase domain, which contains signature motifs found in the nucleotidyl transferase superfamily.     

Synthesis of oligonucleotide derivatives containing a bis-pyrene residue in the main chain.

The oligonucleotide having the bis-pyrene residue in the main chain was synthesized. The preparation of the bis-pyrene was started from the conversion of 2,2-bis-(bromomethyl)-1,3-propanediol into the protected bis-amino derivative. The reaction of the bis-amino derivative with 1-pyrenebutyric acid using DCC/HOBT afforded the desired bis-pyrene. This compound was then converted to the protected phosphormidite. The oligonucleotides possessing the bis-pyrene were synthesized by using the amidite. The oligonucleotides having the bis-pyrene residue can bind to DNA sequence in an aqueous solution to give the duplex with comparable thermal stability as that of the unmodified DNA/DNA duplex. The significantly enhanced pyrene-excimer fluorescence was observed upon hybridization of the bis-pyrene modified oligonucleotides with DNA.     

Binding of MutS protein to oligonucleotides containing a methylated or an ethylated guanine residue, and correlation with mutation frequency

The MutS-based mismatch repair (MMR) system has been conserved from prokaryotes to humans, and plays important roles in maintaining the high fidelity of genomic DNA. MutS protein recognizes several different types of modified base pairs, including methylated guanine-containing base pairs. Here, we looked at the relationship between recognition and the effects of methylating versus ethylating agents on mutagenesis, using a MutS-deficient strain of E. coli. We find that while methylating agents induce mutations more effectively in a MutS-deficient strain than in wild-type, this genetic background does not affect mutagenicity by ethylating agents. Thus, the role of E. coli MMR with methylation-induced mutagenesis appears to be greater than ethylation-induced mutagenesis. To further understand this difference an early step of repair was examined with these alkylating agents. A comparison of binding affinities of MutS with O⁶-alkylated guanine base paired with thymine, which could lead to transition mutations, versus cytosine which could not, was tested. Moreover, we compared binding of MutS to oligoduplexes containing different base pairs; namely, O⁶-MeG:T, O⁶-MeG:C, O⁶-EtG:T, O⁶-EtG:C, G:T and G:C. Dissociation constants (K_d), which reflect the strength of binding, followed the order G:T- > O⁶-MeG:T- > O⁶-EtG:T- = O⁶-EtG:C- ≥ O⁶-MeG:C- > G:C. These results suggest that a thymine base paired with O⁶-methyl guanine is specifically recognized by MutS and therefore should be removed more efficiently than a thymine opposite O⁶-ethylated guanine. Taken together, the data suggest that in E. coli, the MMR system plays a more significant role in repair of methylation-induced lesions than those caused by ethylation.     

Anticonvulsants containing the N-(3-aryl-2-propenoyl) amido pharmacophore

A series of 1-(3-aryl-2-propenoyl)-4-oxopiperidines (1) as well as some related semicarbazones (2) and thiosemicarbazones (3) were prepared in order to determine whether the relative locations of aryl rings and amidic groups would lead to novel anticonvulsant agents. Initially the compounds were administered intraperitoneally to mice and examined in the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and neurotoxicity (NT) screens. The biodata revealed that anticonvulsant properties were displayed by most of the compounds in series (1), in half of the semicarbazones (2) while protection was absent by members of series (3). Molecular modeling was utilized in order to compare the positions of a phenyl ring in relation to amidic groups in representative compounds in series (1-3) with previously reported anticonvulsant agents. Molecular simplification of 4-oxo-1-(3-phenyl-2-propenoyl)piperidine (la) led to 1-(3-phenyl-2-propenoyl)piperidine (7) and N,N-diethylcinnamamide (8) with retention of anticonvulsant properties. Both (la) and (8) afforded protection in the hippocampal kindling screen in rats. When administered orally to rats, (la) and (8) demonstrated activity in the MES screen and in the case of (8), a huge protection index was observed revealing it to be an important lead compound. The IC50 values of all of the compounds towards murine P388 cells were in excess of 50 microM while several compounds displayed cytotoxicity towards Mycobacterium tuberculosis.     

Synthesis of a glycotripeptide and a glycosomatostatin containing the 3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-serine residue

3-O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-N- (tert-butyloxycarbonyl)-L-serine was synthesized and condensed by the solid-phase procedure to give the sequence Gly-[beta-D-GlcpNAc-(1 leads to 3)-Ser]-Ala-OH and beta-D-GlcpNAc-(1 leads to 3)-Ser-13-somatostatin. The synthetic glycopeptides appeared homogeneous on t.l.c. and l.c. examination and showed the correct amino acid composition and 2-amino-2-deoxy-D-glucose content. The structure of Gly-[beta-D-GlcpNAc-(1 leads to 3)-Ser]-Ala-OH was further confirmed by mass spectrometry of the N-acetyl permethyl derivative, and by n.m.r. spectroscopy.     

Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA)

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5′- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5′-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.     

Chemical synthesis of oligonucleotides containing (M+4) guanine

HPLC MS/MS has shown great potential in the measurement of DNA oxidative damage. Its accuracy depends on the use of multiply isotopically labelled internal standards. In this report, multiply isotopically labelled (M + 4) guanine internal standards were prepared in the form of base, nucleoside, as well as DNA oligomer. To our knowledge, this is the first chemical synthesis of oligomers containing (M + 4) guanine, and we believe that they can be used to develop a procedure that can make further improvement to the existing analytical procedures.     

Group separation of peptides by ligand-exchange chromatography with a Sephadex containing N-(2-pyridylmethyl)glycine

The synthesis of 2-hydroxy-3[N-(2-pyridylmethyl)glycine]propyl Sephadex ether--a new chelating resin--is described. This resin has been employed in the form of its Cu2+ complex to separate peptides, as a group, from alpha-amino acids and NaCl. Ninety-seven ligands of different structures were separated chromatographically at room temperature. It was shown that two structural parameters of the ligands control the separation process, namely, the presence of ligand donor groups and the possibility for them to form chelate rings of suitable size. Separation of peptides from alpha-, gamma-, delta-amino acids, N-acetyl derivatives of amino acids (except N-Ac-Trp), and NaCl is possible if the peptides fulfill the following structural requirements: the peptide molecule must have a free terminal amino group; a carbonyl group (of the peptide linkage) must be situated in the alpha- or beta-position of the free amino group; and the peptide may not contain an imidazole residue (except Gly-Gly-His). A relationship was found between the log k' and the corresponding pKHHL, log KCuCuL, and log KCuBipyCuBipyL values. Interpretation of the different K' values was possible based on the different basicities of the terminal amino groups and on the structures of the different side chains of the peptides.     

Analgesic dipeptide derivatives. II. Synthetic dipeptides containing a C-terminal 2-(2-nitrophenylsulfenyl) tryptophan residue

The syntheses of dipeptide derivatives of the general formula X-Trp(Nps)-OR (R = H or Me; X = Lys, Gly, Ala, Leu, Ser, Glu, His, Arg, Orn, Dpr, Gpr and Har) are described. The analgesic activities of Gpr- and Har-containing dipeptides have been evaluated and, in the light of these results, the influence of the side chain length of the basic amino acid on the analgesic effect is studied.     

Structure and conformation of the potent antiherpes agent 9-(2-hydroxyethoxymethyl) guanine (acycloguanosine)

The structure and conformation of 9-(2-hydroxyethoxymethyl)guanine (Acyclovir), a potent inhibitor of herpes simplex viruses, was determined by X-ray diffraction. The asymmetric unit contains three independent molecules of the nucleoside analogue and two molecules of water. In two of them the acyclic chain at N(9) is partly in the folded form; in the third it is fully extended. Both conformations resemble that of guanosine, and the conformation of all three molecules about the glycosidic bond is in the high anti range. The relevance of the conformational data to the substrate susceptibility of Acyclovir towards kinases, and to its antiviral activity, is examined.     

The solution structure of an oligonucleotide duplex containing a 2'-deoxyadenosine-3-(2-hydroxyethyl)- 2'-deoxyuridine base pair determined by NMR and molecular dynamics studies

Determination of the solution structure of the duplex d(GCAAGTC(HE)AAAACG)·d(CGTTTTAGACTTGC) containing a 3-(2-hydroxyethyl)-2′-deoxyuridine·deoxyadenine (HE·A) base pair is reported. The three-dimensional solution structure, determined starting from 512 models via restrained molecular mechanics using inter-proton distances and torsion angles, converged to two final families of structures. For both families the HE and the opposite A residues are intrahelical and in the anti conformation. The hydroxyethyl chain lies close to the helix axis and for one family the hydroxyl group is above the HE·A plane and in the other case it is below. These two models were used to start molecular dynamic calculations with explicit solvent to explore the hydrogen bonding possibilities of the HE·A base pair. The dynamics calculations converge finally to one model structure in which two hydrogen bonds are formed. The first is formed all the time and is between HEO4 and the amino group of A, and the second, an intermittent one, is between the hydroxyl group and the N1 of A. When this second hydrogen bond is not formed a weak interaction CH···N is possible between HEC7H2 and N1A21. All the best structures show an increase in the C1′–C1′ distance relative to a Watson–Crick base pair.     

Reactive oligonucleotide derivatives containing methylphosphonate groups. V. Increased effectiveness of complementary addressed modification of target DNA by alkylating derivatives of methylphosphonate analogs of octathymidylate containing N-(2-hydroxyethyl)phenazinium residues

Efficiency of the intracomplex alkylation of octadecadeoxyribonucleotide d(pC5A8C5) (target) by Rp- and Sp-individual diastereomers of the methylphosphonate octathymidylate 4-(N-methyl-N-2-chloroethylamino)benzyl phosphoramide (-pNHCH2RCl) derivatives bearing an additional N-(2-hydroxyethyl)phenazinium residue (phn), viz. ClRCH2NHpTp.(TpTp)3TpNH(CH2)2NHPhn (I) and PhnNH(CH2)2NHpTp(TpTp)3TpNHCH2RCl (II) (p = -OP(O) (CH3)O-), has been investigated. Stabilisation of the complementary complex formed by the target oligonucleotide and methylphosphonate oligonucleotide derivatives by the Phn group considerably rose the efficiency of the intracomplex alkylation of the target as compared with alkylation by reagents without Phn. RP-isomeric derivatives of (I) and (II) proved to be the most effective alkylating reagents. Specificity of alkylation of nucleic acid target by reagents (I) and (II) is studied.     

Structure and dynamics of a DNA duplex containing single alpha-anomeric deoxyadenosine residue.

Structure and dynamics of an undecamer DNA duplex containing a single alpha-anomeric deoxyadenosine residue placed in opposition to a thymidine unit have been studied using simulation of molecular dynamics in aqueous solution. Despite several noticeable deviations from the B-DNA duplex structure caused by the anomerisation, such as: West type puckering of the alpha-anomeric sugar, disrupted base stacking pattern and unstable duplex bending, the formation of a non-classical alpha-dA-T pair was observed. A novel way of visual presentation of trajectory data allowing high throughput screening of the conformational parameters is presented.     

Synthesis and recognition by DNA polymerases of a reactive nucleoside, 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide

We report the synthesis of a new nucleoside, 1-(2-deoxy-beta-D-erythro-pentofuranosyl)-imidazole-4-hydrazide (dY(NH2)) as a reactive monomer for DNA diversification. The 5'-triphosphate derivative (dY(NH2)TP, 1) was evaluated in vitro as a substrate for several DNA polymerases. Primer extension reactions showed that dYNH2TP was well tolerated by KF (exo(-)) and Vent (exo-) DNA polymerases. One dYNH2MP was incorporated opposite each canonical base with an efficiency depending on the template base (A approximately T > G > C). Significant elongation after YNH2 incorporation was observed independently of the YNH2:N base pair formed. When the nucleobase YNH2 was incorporated into synthetic oligodeoxynucleotides via the phosphoramidite derivative 11, it directed the insertion of natural bases as well as itself. The mutagenicity of dYNH2TP was evaluated by PCR amplification using Vent (exo-) DNA polymerase. The triphosphate dY(NH2)TP was preferentially incorporated as a dATP or dGTP analogue and led to misincorporations at frequencies of approximately 2 x 10(-2) per base per amplification. A high proportion of transversions with a large distribution of all possible mutations was obtained. The reactivity of the nucleobase YNH2 within a template with several aldehydes was demonstrated.