Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5'-d(CGCXAATTYGCG)-3'.

The nucleoside analogs 1-(2'-deoxy-beta-D-ribofuranosyl)- 3-nitropyrrole (9), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2'-deoxy-beta-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2'-deoxy-beta-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5'-d(CGCXAATTYGCG)-3'in the fourth position from the 5'-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3'-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded T m values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which T m values differed by 5 degrees C and Delta G 25 degrees C ranged from -6.1 to -6.5 kcal/mol. 5-Nitroindole gave duplexes with significantly higher thermal stability, with Tm values varying from 35.0 to 46.5 degrees C and -Delta G 25 degrees C ranging from 7.7 to 8.5 kcal/mol. Deoxyinosine (22), a natural analog which has found extensive use as a universal nucleoside, is far less non-discriminating than any of the nitroazole derivatives. Tm values ranged from 35.4 degrees C when paired with G to 62.3 degrees C when paired with C. The significance of the nitro substituent was determined by comparison of the base pairing properties of a simple azole nucleoside, 1-(2'-deoxy-beta-D-ribofuranosyl)pyrazole (12). The pyrazole-containing sequences melt at 10-20 degrees C lower than the corresponding nitropyrazole-containing sequences. On average, the pyrazole-containing sequences were equivalent in stability (average Delta G = -4.8 kcal/mol) to the sequences containing an abasic site (average Delta G = -4.7 kcal/mol).     

The 6-thioguanine/5-methyl-2-pyrimidinone base pair.

As part of a program to determine the physical possibility of expanding the number of types of base pairs in DNA, the pairing stabilities of the analog bases 6-thioguanine (GS) and 5-methyl-2-pyrimidinone (TH) in oligodeoxynucleotides were measured. Procedures were developed to synthesize oligodeoxynucleotides with the analog bases. The sequences of the synthesized oligomers were T-C-G-A-C-G-G-X-Y-C-C-G. An enzymatic procedure was developed to measure relative association constants of oligomer pairs with the self complementary reference oligomer, X = A and Y = T, K(T/A) = K. The results were K(C/G) = (5 +/- .5)K, K(TH/GS) = K/(1 +/- .5), K(T/G) = K/(9 +/- 3), K(TH/G) = K/(25 +/- 5), K(C/GS) less than K/30, K(TH/A) less than K/40, K(T/GS) less than K/40, K(C/A) less than K/40. The results with the standard bases are consistent with other methods of measurement. The stability of the base pair GS/TH is approximately the same as the standard base pair A/T.     

Stable isotope labeling-mass spectrometry analysis of methyl- and pyridyloxobutyl-guanine adducts of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in p53-derived DNA sequences

The p53 tumor suppressor gene is a primary target in smoking-induced lung cancer. Interestingly, p53 mutations observed in lung tumors of smokers are concentrated at guanine bases within endogenously methylated (Me)CG dinucleotides, e.g., codons 157, 158, 245, 248, and 273 ((Me)C = 5-methylcytosine). One possible mechanism for the increased mutagenesis at these sites involves targeted binding of metabolically activated tobacco carcinogens to (Me)CG sequences. In the present work, a stable isotope labeling HPLC-ESI(+)-MS/MS approach was employed to analyze the formation of guanine lesions induced by the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) within DNA duplexes representing p53 mutational "hot spots" and surrounding sequences. Synthetic DNA duplexes containing p53 codons 153-159, 243-250, and 269-275 were prepared, where (Me)C was incorporated at all physiologically methylated CG sites. In each duplex, one of the guanine bases was replaced with [1,7,NH(2)-(15)N(3)-2-(13)C]-guanine, which served as an isotope "tag" to enable specific quantification of guanine lesions originating from that position. After incubation with NNK diazohydroxides, HPLC-ESI(+)-MS/MS analysis was used to determine the yields of NNK adducts at the isotopically labeled guanine and at unlabeled guanine bases elsewhere in the sequence. We found that N7-methyl-2'-deoxyguanosine and N7-[4-oxo-4-(3-pyridyl)but-1-yl]guanine lesions were overproduced at the 3'-guanine bases within polypurine runs, while the formation of O(6)-methyl-2'-deoxyguanosine and O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]-2'-deoxyguanosine adducts was specifically preferred at the 3'-guanine base of 5'-GG and 5'-GGG sequences. In contrast, the presence of 5'-neighboring (Me)C inhibited O(6)-guanine adduct formation. These results indicate that the N7- and O(6)-guanine adducts of NNK are not overproduced at the endogenously methylated CG dinucleotides within the p53 tumor suppressor gene, suggesting that factors other than NNK adduct formation are responsible for mutagenesis at these sites.     

Novel ring transformation of 5-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-isoxazole-4-carbaldehyde with 1,2-diaminobenzenes to 3-cyano-1,5-benzodiazepine C-nucleosides

Syntheses of 3-cyano-7- and 8-substituted-4-(beta-D-ribofuranosyl)-1H-1,5-benzodiazepines were reported. Treatment of isoxazole carbaldehyde with 1,2-diamino-4-nitrobenzene in chloroform gave a Schiffs base, 4-(2-amino-5-nitrophenyl)iminomethyl-5-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)isoxazole in 82% yield with no trace of the other regioisomer. The cyclocondensation of the resulting Schiffs base in benzene containing trifluoroacetic acid (TFA) gave 3-cyano-8-nitro-4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1H-1,5-benzodiazepine in 49% yield. The same reaction of isoxazole carbaldeyde with 1,2-diamino-4-methoxy- and 4-chlorobenzenes afforded the corresponding Schiffs bases. Extending the reaction time for Schiffs base gave the corresponding cyanobenzodiazepines in good yields. Debenzoylation of the compounds with sodium methoxide produced deprotected C-nucleosides.     

Chemical synthesis and properties of (6-4) photoproduct and its analogs.

We report the preparation of a deoxyribooligonucleotide containing a new thymine (6-4) photoproduct analog. The (6-4) photoproduct is one of the major forms of DNA lesions, and leads to mutation in DNA. An antibody (64M5) that binds the (6-4) photoproduct has been described. To investigate the interaction of the photoproduct with the 64M5 antibody, we prepared a (6-4) photoproduct analog in which the two thymines were connected with a formacetal linkage. With UV-irradiation, the thymine dimer with the formacetal linkage reacted to the (6-4) photoproduct faster than the phosphodiesterified dimer, and the yields of the analog was higher than those of the natural thymine dimer. The 64M5 antibody exhibited sufficient binding to a tetranucleotide containing the (6-4) photoproduct analog with a formacetal linkage, although the association constant was slightly lower than that for the natural lesion. This (6-4) photoproduct analog may be useful for investigation of other proteins that recognize the (6-4) photoproduct.     

Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog

Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2′-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base.     

Cyclohexenyl Nucleosides and Related Compounds

Abstract

Cis and trans-1-(4-hydroxy-2-cyclohexenyl)- and 1-(2-hydroxy-5-cyclohexenyl) thymines were obtained by stereospecific routes. Oxidation of the 1, 4-products afforded 1-(4-oxo-2-cyclohexenyl)thymine, the carbocyclic analog of a reportedly antiviral ketopyranosyl nucleoside. Exclusive 1, 6-conjugate addition occurred with heterocyclic bases and methyl 1, 3-cyclohexadiene-1-carboxylate. Reduction of the thymine adduct gave 1-(4-hydroxymethyl1-3-cyclohexenyl)thymine. Michael-type addition provided a direct route to 3-oxocycloalkyl nucleosides, and lactone nucleosides resulted from addition of bases to α-methylene-γ-butyrolactone. Anti-HIV screening revealed no activity for the new compounds.     

Inhibition of DNA repair glycosylases by base analogs and tryptophan pyrolysate, Trp-P-1

DNA base analogs, 2,4,5,6-substituted pyrimidines and 2,6-substituted purines were tested as potential inhibitors of E. coli Fpg protein (formamidopyrimidine -DNA glycosylase). Three of the seventeen compounds tested revealed inhibitory properties. 2-Thioxanthine was the most efficient, inhibiting 50% of 2,6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine (Fapy-7MeG) excision activity at 17.1 microM concentration. The measured K(i) was 4.44 +/- 0.15 microM. Inhibition was observed only when the Fpg protein was first challenged to its substrate followed by the addition of the base analog, suggesting uncompetitive (catalytic) inhibition. For two other compounds, 2-thio- or 2-oxo-4,5,6-substituted pyrimidines, IC(50) was only 343.3 +/- 58.6 and 350 +/- 24.4 microM, respectively. No change of the Fpg glycosylase activity was detected in the presence of Fapy-7MeG, up to 5 microM. We also investigated the effect of DNA structure modified by tryptophan pyrolysate (Trp-P-1) on the activity of base excision repair enzymes: Escherichia coli and human DNA glycosylases of oxidized (Fpg, Nth) and alkylated bases (TagA, AlkA, and ANPG), and for bacterial AP endonuclease (Xth protein). Trp-P-1, which changes the secondary DNA structure into non-B, non-Z most efficiently inhibited excision of alkylated bases by the AlkA glycosylase (IC(50) = 1 microM). The ANPG, TagA, and Fpg proteins were also inhibited although to a lesser extent (IC(50) = 76.5 microM, 96 microM, and 187.5 microM, respectively). Trp-P-1 also inhibited incision of DNA at abasic sites by the beta-lyase activity of the Fpg and Nth proteins, and to a lesser extent by the Xth AP endonuclease. Thus, DNA conformation is critical for excision of damaged bases and incision of abasic sites by DNA repair enzymes.     

Homo-C-nucleoside analogs III. Studies on the base-catalyzed dehydrative cyclization of 4-(d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole

Treatment of 4-(d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole with one molar equivalent of 2,4,6-triisopropylbenzenesulfonyl chloride (TIBSCl) in pyridine solution afforded the homo-C-nucleoside analog; 4-(2,5-anhydro-d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole in 54% yield and 4-(α-d-arabinopyranosyl)-2-phenyl-2H1,2,3-triazole analog in 3% yield. The 4-(5-O-triisopropylbenzenesulfonyl)-d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole analog was isolated as an intermediate and identified as its tetra-O-acetyl derivative. The 4-(5-chloro-5-deoxy-d-manno-pentitol-1-yl)-2-phenyl-2H-1,2,3-triazole analog was isolated as a byproduct. The structure and anomeric configuration of the products were determined by acylation, NMR spectroscopy, and mass spectrometry.     

Human P1-450 gene sequence and correlation of mRNA with genetic differences in benzo[a]pyrene metabolism.

The human P1-450 gene (6,311 base pairs), as well as the 5' (1,604 bases) and 3' (113 bases) flanking regions, have been completely sequenced. Four highly homologous boxes (61, 82, 56 and 97 base pairs) between the human and mouse P1-450 genes are found in the "TATA" box promoter region, -226, -338, and -450 upstream from the cap site, respectively. Nine genomic-DNA samples were digested with each of 23 restriction endonucleases and probed with human P1-450 cDNA fragments; restriction fragment length polymorphisms are detected, although it remains to be seen whether such a recombinant DNA test will be useful in determining individuals at increased risk for cigarette smoking-induced cancer and toxicity. We show in this report, however, that human inducible P1-450 mRNA concentrations are very highly correlated (r = 0.98; N = 6) with genetic differences in benzo[a]pyrene metabolism in mitogen-activated lymphocyte cultures.     

5-Nitrouridine-monohydrate: crystal structure and conformation.

The crystal structure of 5-nitrouridine was determined by X-ray analysis. The pyrimidine ring is slightly non-planar, showing a shallow boat conformation. The nitro group has no influence on the C4 - O4 bond length as compared to uridine. The ribose shows the C3'-endo conformation and the base is in the anti orientation to the sugar with a torsion angle of 25.6 degrees. This conformation is stabilized by a hydrogen bond from the base to the ribosyl moiety (H6 ... 05'). Stacking interactions between neighboring bases are almost negligible in the crystal. A water molecule is involved in a bifurcated donating hydrogen bond to 04 and to 052 of the nitro group of the one base and an accepting bond from the H3 of the other base. Two more hydrogen bonds are formed between the water molecule and the ribose. The structural aspects of 5-nitrouridine are discussed with respect to the special stacking features found for 5-nitro-1-(beta-D-ribosyluronic acid)-uracil monohydrate in the crystal (1).     

Crystal structure of the 64M-2 antibody Fab fragment in complex with a DNA dT(6-4)T photoproduct formed by ultraviolet radiation

DNA photoproducts with (6-4) pyrimidine-pyrimidone adducts formed by ultraviolet radiation are implicated in mutagenesis and cancer, particularly skin cancer. The crystal structure of the Fab fragment of the murine 64M-2 antibody specific to DNA T(6-4)T photoproducts is determined as a complex with dT(6-4)T, a (6-4) pyrimidine-pyrimidone photodimer of dTpT, at 2.4 A resolution to a crystallographic R-factor of 0.199 and an R(free) value of 0.279. The 64M-2 Fab molecule is in an extended arrangement with an elbow angle of 174 degrees, and its five complementarity-determining regions, except L2, are involved in the ligand binding. The bound dT(6-4)T ligand adopting a ring structure with (6-4) linked 5' thymine-3' pyrimidone bases is fully accommodated in an antigen-binding pocket of about 15 Ax10 A. The 5'-thymine and 3'-pyrimidone bases are in half-chair and planar conformations, respectively, and are nearly perpendicular to each other. The 5'-thymine base is hydrogen-bonded to Arg95H and Ser96H, and is in van der Waals contact with Tyr100iH. The 3'-pyrimidone base is hydrogen-bonded to His35H, and is in contact with Trp33H. Three water molecules are located at the interface between the bases and the Fab residues. Hydrogen bonds involving these water molecules also contribute to Fab recognition of the dT(6-4)T bases. The sugar-phosphate backbone connecting the bases is surrounded by residues His27dL, Tyr32L, Ser92L, Trp33H, and Ser58H, but is not hydrogen-bonded to these residues.     

Structural and conformational studies on deoxyguanosyl-3',5'-deoxyadenosine monophosphate and its ethyl phosphotriester analogs--left-handed dimers

The mode of base-base stacking, the handedness and the sugar(dGpA)phosphate backbone conformation of deoxyguanosyl 3'-5' deoxyadenosine and its diastereomeric ethyl phosphotriester analogs were studied by 1H NMR, UV and CD spectroscopy. The results indicate the three dimers are left-handed, while the sugar phosphate backbone is comprised predominantly of C2-endo,gg(C4-C5) and g'g (C5-O) conformers. The two bases are extensively stacked and interact about 90 degrees along the dyad axes. The extent of base overlap in dGpA is slightly greater than in either ethyl phosphotriester analog. The absolute configurations of the two ethyl phosphotriester diastereoisomers of dGpA can be assigned by one-dimensional and two-dimensional 1H NMR nuclear Overhauser enhancement experiments.     

Structural similarities and differences between H1- and H2-family DNA minihairpin loops: NMR studies of octameric minihairpins

TheDNA sequences 5′-d(CGC-AC-GCG)-3′ (HPAC), 5′-d(CGC-AA-GCG)-3′ (HPAA), 5′-d(CGC-TC-GCG)-3′ (HPTC), and 5′-d(CGC-CT-GCG)-3′ (HPCT), were studied by means of nmr spectroscopy. At low DNA concentration and no added salt all four molecules adopt a minihairpin structure, containing three Watson–Crick base pairs and a two-residue loop. The structure of the HPAC hairpin is based on quantitative distance restraints, derived by a full relaxation matrix approach (iterative relaxation matrix approach), together with torsion angles obtained from coupling constant analysis. The loop folding is of the H1-family type, characterized by continuous 3′-5′ stacking of the loop bases on the duplex stem. The structure of the HPAA hairpin is similar to that of HPAC, but is more flexible and has a lower thermodynamic stability (T_m 326 K vs 320 K). According to “weakly” distance-constrained simulations in water on the HPAC minihairpin, the typical H1-family loop folding remains intact during the simulation. However, residue-based R factors of simulated nuclear Overhauser effect spectroscopy spectra, free molecular dynamics simulations in vacuo, and unusual chemical shift profiles indicate partial destacking of the loop bases at temperatures below the overall melting midpoint. The dynamic nature of the loop bases gives insight into the geometrical tolerances of stacking between bases in H1-family minihairpin loops. The HPTC and HPCT minihairpins, both containing a pyrimidine base at the first position in the loop, adopt a H2-family type folding, in which the first loop base is loosely bound in the minor groove and the second loop base is stacked upon the helix stem. The thermal stability for these two hairpins corresponds to 327–329 K, but depends on local base sequence. Preference for the type of folding depends on a single substitution from a pyrimidine (H2 family) to a purine (H1 family) at the first position of the miniloop and is explained by differences in base stacking energies, steric size, and the number of possible candidates for hydrogen bonds in the minor groove. In view of newly collected data, previous models of the H1-family and H2-family hairpins had to be revised and are now compatible with the reported HPTC and HPAC structures. The structural difference between the refined structure of HPAC and HPTC show that a conversion between H1-family and H2-family hairpins is geometrically possible by a simple pivot point rotation of 270° along two torsion angles, thereby swiveling the first loop base from a stacked position in a H1-family folding toward a position in the minor groove in a H2-family folding. The second loop residue subsequently shifts to the position of the first base in a concerted fashion. © 1998 John Wiley & Sons, Inc. Biopoly 46: 375–393, 1998     

2''-Deoxy-3,7-dideazaguanosine and related compounds. Synthesis of 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl) and 1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]pyridin-4(5H)-one via direct glycosylation of a pyrrole precursor.

The synthesis of two new analogs of 2'-deoxyguanosine, 6-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H-pyrrolo[3,2-c] pyridin-4(5H)-one (8) and 6-amino-1-beta-D-arabinofuranosyl-1H-pyrrolo[3,2-c]-pyridin-4(5H)-one (13) has been accomplished by glycosylation of the sodium salt of ethyl 2-cyanomethyl-1H-pyrrole-3-carboxylate (4c) using 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose( 5) and 1-chloro-2,3,5-tri-O-benzyl-alpha-D-arabinofuranose (9), respectively. The resulting blocked nucleosides, ethyl 2-cyanomethyl-1-(2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro- pentofuranosyl)-1H-pyrrole-3-carboxylate (6) and ethyl 2-cyanomethyl-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)- 1H-pyrrole-3-carboxylate, were ring closed with hydrazine to form 5-amino-6-hydrazino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-1H- pyrrolo[3,2-c]-pyridin-4(5H)-one (7) and 5,6-diamino-1-(2,3,5-tri-O-benzyl-beta-D-arabinofuranosyl)-1H- pyrrolo[3,2-c]pyridin-4(5H)-one (11), respectively. Treatment of 7 with Raney nickel provided the 2'-deoxyguanosine analog 8 while reaction of 11 with Raney nickel followed by palladium hydroxide/cyclohexene treatment gave the 2'-deoxyguanosine analog 13. The anomeric configuration of 8 was assigned as beta by proton NMR, while that of 13 was confirmed as beta by single-crystal X-ray analysis of the deblocked precursor ethyl 2-cyanomethyl-1-beta-D-arabinofuranosyl-1H-pyrrole-3-carboxylate (10a).     

Direct determination of uracil in [32P,uracil-3H]poly(dA.dT) and bisulfite-treated phage PM2 DNA

A simple but effective technique for determining the presence of uracil existing as either A:U base pairs or G:U base pairs in DNA was developed. DNA is degraded to deoxynucleoside 3'-monophosphates by a combination of micrococcal nuclease and spleen phosphodiesterase. The monophosphates are converted to 5'-end-labeled 32P-labeled diphosphates in a reaction catalyzed by T4 polynucleotide kinase. The resultant product is then converted to 5'-end-labeled deoxynucleoside monophosphates by P1 nuclease digestion, which specifically removes 3'-phosphates. Successful separation of labeled dUMP from conventional bases in DNA is achieved by two-dimensional polyethyleneimine chromatography, with its detection determined by autoradiography and liquid scintillation counting. The sensitivity of the technique described can detect a minimum 1 X 10(-16) mol of dUMP in DNA. Additionally, the detection of 5-methylcytosine in placental DNA demonstrates the flexibility of the technique for the analysis of modified bases in DNA.     

AarI,a restriction endonuclease from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic sequence 5'-CACCTGC(N)4/8-3'

A new type II restriction endonuclease AarI has been isolated from Arthrobacter aurescens SS2-322. AarI recognizes the non-palindromic heptanucleotide sequence 5'-CACCTGC(N)4/8-3' and makes a staggered cut at the fourth and eighth bases downstream of the target duplex producing a four base 5'-protruding end. AarI activity is stimulated by oligodeoxyribonucleotide duplexes containing an enzyme-specific recognition sequence.     

Human DNA primase uses Watson-Crick hydrogen bonds to distinguish between correct and incorrect nucleoside triphosphates

Human DNA primase synthesizes short RNA primers that DNA polymerase alpha further elongates. Primase readily misincorporates the natural NTPs and will generate a wide variety of mismatches. In contrast, primase exhibited a remarkable resistance to polymerizing NTPs containing unnatural bases. This included bases whose shape was almost identical to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base [e.g., 5- and 6-(trifluoromethyl)benzimidazole], bases much more hydrophobic than a natural base [e.g., 4- and 7-(trifluoromethyl)benzimidazole], bases of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-D-guanine), and bases capable of forming only one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). Primase only polymerized NTP analogues containing bases capable of forming hydrogen bonds between the equivalent of both N-1 and the exocyclic group at C-6 of a purine NTP (2-fluoroadenine, 2-chloroadenine, 3-deazaadenine, and hypoxanthine) and N-3 and the exocyclic group at C-4 of a pyrimidine. These data indicate that human primase requires the formation of Watson-Crick hydrogen bonds in order to polymerize a NTP, a situation very different than what is observed with some DNA polymerases. The implications of these results with respect to current theories of how polymerases discriminate between right and wrong (d)NTPs are discussed.     

4-Hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl]piperidines: selective h5-HT1D agonists for the treatment of migraine

A series of 4-hydroxy-1-[3-(5-(1,2,4-triazol-4-yl)-1H-indol-3-yl)propyl] piperidines was investigated as potential selective h5-HT1D agonists for the treatment of migraine. The 4-[(N-benzyl-N-methyl)amino]methyl analog 12a was found to be a full agonist at the h5-HT1D receptor with good binding selectivity over the h5-HT1B receptor.     

A non-nucleotide-based linking method for the preparation of psoralen-derivatized methylphosphonate oligonucleotides.

A method is reported for conjugating an analog of 4'-(aminomethyl)-4,5',8- trimethylpsoralen to methylphophonate oligonucleotides. This method enables the psoralen moiety to be coupled to the phosphonate backbone between any two desired bases in a sequence. When hybridized to a target mRNA, the psoralen moiety can be directed toward a uridine base and, in turn, can undergo a photo-addition reaction with the target under UV irradiation at 365 nm. Several different non-nucleotide-based amino-linker reagents have been prepared for incorporation into methylphosphonate oligonucleotides by standard phosphonamidite chemistry. In addition, an N-hydroxysuccinimide activated ester analog of 4'-[(3-carboxypropionamido)methyl]-4,5',8- trimethylpsoralen has been synthesized for conjugation to the amino-linker moieties. Using this approach, we have prepared a number of psoralen-methylphosphonate-oligonucleotide conjugates which are complementary to the chimeric bcr/abl mRNA associated with chronic myelogenous leukemia. Solution hybridization studies with a 440-base subfragment of the bcr/abl RNA have shown that the psoralen moiety does not adversely affect duplex stability. Polyacrylamide gel electrophoresis analyses have demonstrated that the psoralen-oligonucleotide conjugates undergo photo-addition to the RNA in a sequence-specific manner. Optimal photo-addition occurs when the psoralen moiety is inserted adjacent to one or more adenine residues in the oligonucleotide sequence, particularly between adenine and thymine (5'-3'). This internal labeling approach greatly increases the number of potential target sites available for photo-cross-linking experiments.