Synthesis of 6-(2-thienyl)purine nucleoside derivatives that form unnatural base pairs with pyridin-2-one nucleosides

Unnatural bases, 2-amino-6-(2-thienyl)purine and 2-amino-6-(2-furanyl)purine, were newly designed to replace the previously developed purine analogue, 2-amino-6-(N,N-dimethylamino)purine, which specifically pairs with pyridin-2-one. These nucleoside derivatives were synthesized via the 6-substitution of 6-iodopurine nucleosides with tributylstannylthiophene or tributylstannylfuran. As compared with 2-amino-6-(N,N-dimethylamino)purine, 2-amino-6-(2-thienyl)purine reduced the interference in the stacking interactions with the neighboring bases in a DNA duplex and improved the efficiency of the enzymatic incorporation of the nucleoside triphosphate of pyridin-2-one opposite the unnatural base.     

Cognate base‐pair selectivity of hydrophobic unnatural bases in DNA ligation by T4 DNA ligase

We present cognate base pair selectivity in template‐dependent ligation by T4 DNA ligase using a hydrophobic unnatural base pair (UBP), Ds‐Pa. T4 DNA ligase efficiently recognizes the Ds‐Pa pairing at the conjugation position, and Ds excludes the noncognate pairings with the natural bases. Our results indicate that the hydrophobic base pairing is allowed in enzymatic ligation with higher cognate base‐pair selectivity, relative to the hydrogen‐bond interactions between pairing bases. The efficient ligation using Ds‐Pa can be employed in recombinant DNA technology using genetic alphabet expansion, toward the creation of semi‐synthetic organisms containing UBPs.     

Unnatural base pairs between 2-amino-6-(2-thienyl)purine and the complementary bases.

The unnatural base, 2-amino-6-(2-thienyl)purine (designated as s), instead of 2-amino-6-(N,N-dimethylamino)purine (designated as x), was designed in order to improve the specificity and efficiency of the base pairing with pyridin-2-one (designated as y). DNA fragments containing s were chemically synthesized, and the thermal stability and the enzymatic reactions involving the s-y pairing were examined. Thermal denaturation experiments showed that the DNA duplex (12-mer) containing the s-y pair was more stable than that containing the x-y pair. The incorporation of dyTP was also more advantageous to the s-y pairing than the x-y pairing in single-nucleotide insertion experiments using the Klenow fragment of Escherichia coli DNA polymerase I.     

An unnatural hydrophobic base, 4-propynylpyrrole-2-carbaldehyde, as an efficient pairing partner of 9-methylimidazo[(4,5)-b]pyridine

To develop unnatural base pairs that function in replication, we designed 4-propynylpyrrole-2-carbaldehyde (designated as Pa′) and synthesized the nucleoside derivatives of Pa′. The base pairing of Pa′ with the partner, 9-methylimidazo[(4,5)-b]pyridine (Q), was compared to that of pyrrole-2-carbaldehyde (Pa), which was previously developed as a specific pairing partner of Q. The thermal stability of a DNA duplex containing the Q–Pa′ pair and the incorporation efficiency of the Pa′ substrate (dPa′TP) into DNA opposite Q by the Klenow fragment of Escherichia coli DNA polymerase I were improved, in comparison with those of the Q–Pa pair. These improvements result from the increased hydrophobicity and stacking stability of Pa′ by the introduction of the propynyl group to Pa, providing valuable information for the further development of unnatural base pairs toward the expansion of the genetic alphabet.     

Substituent effects on the pairing and polymerase recognition of simple unnatural base pairs

As part of an effort to develop stable and replicable unnatural base pairs, we have evaluated a large number of unnatural nucleotides with predominantly hydrophobic nucleobases. Despite its limited aromatic surface area, a nucleobase analog scaffold that has emerged as being especially promising is the simple phenyl ring. Modifications of this scaffold with methyl and fluoro groups have been shown to impact base pair stability and polymerase recognition, suggesting that nucleobase shape, hydrophobicity and electrostatics are important. To further explore the impact of heteroatom substitution within this nucleobase scaffold, we report the synthesis, stability and polymerase recognition of nucleoside analogs bearing single bromo- or cyano-derivatized phenyl rings. Both modifications are found to generally stabilize base pair formation to a greater extent than methyl or fluoro substitution. Moreover, polymerase recognition of the unnatural base pairs is found to be very sensitive to both the position and nature of the heteroatom substituent. The results help identify the determinants of base pair stability and efficient replication and should contribute to the effort to develop stable and replicable unnatural base pairs.     

Highly specific unnatural base pair systems as a third base pair for PCR amplification

Toward the expansion of the genetic alphabet of DNA, we present highly efficient unnatural base pair systems as an artificial third base pair for PCR. Hydrophobic unnatural base pair systems between 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds) and 2-nitro-4-propynylpyrrole (Px) were fine-tuned for efficient PCR, by assessing the amplification efficiency and fidelity using different polymerases and template sequence contexts and modified Px bases. Then, we found that some modifications of the Px base reduced the misincorporation rate of the unnatural base substrates opposite the natural bases in templates without reducing the Ds-Px pairing selectivity. Under optimized conditions using Deep Vent DNA polymerase, the misincorporation rate was extremely low (0.005%/bp/replication), which is close to that of the natural base mispairings by the polymerase. DNA fragments with different sequence contexts were amplified ～10(10)-fold by 40 cycles of PCR, and the selectivity of the Ds-Px pairing was >99.9%/replication, except for 99.77%/replication for unfavorable purine-Ds-purine motifs. Furthermore, >97% of the Ds-Px pair in DNA survived in the 10(28)-fold amplified products after 100-cycle PCR (10 cycles repeated 10 times). This highly specific Ds-Px pair system provides a framework for new biotechnology.     

Altered deoxyribonucleotide pools in T-lymphoblastoid cells expressing the multisubstrate nucleoside kinase of Drosophila melanogaster

The multisubstrate nucleoside kinase of Drosophila melanogaster (Dm-dNK) can be expressed in human solid tumor cells and its unique enzymatic properties makes this enzyme a suicide gene candidate. In the present study, Dm-dNK was stably expressed in the CCRF-CEM and H9 T-lymphoblastoid cell lines. The expressed enzyme was localized to the cell nucleus and the enzyme retained its activity. The Dm-dNK overexpressing cells showed approximately 200-fold increased sensitivity to the cytostatic activity of several nucleoside analogs, such as the pyrimidine nucleoside analogs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and 1-beta-d-arabinofuranosylthymine (araT), but not to the antiherpetic purine nucleoside analogs ganciclovir, acyclovir and penciclovir, which may allow this technology to be applied in donor T cells and/or rescue graft vs. host disease to permit modulation of alloreactivity after transplantation. The most pronounced effect on the steady-state dNTP levels was a two- to 10-fold increased dTTP pool in Dm-dNK expressing cells that were grown in the presence of 1 microm of each natural deoxyribonucleoside. Although the Dm-dNK expressing cells demonstrated dNTP pool imbalances, no mitochondrial DNA deletions or altered mitochondrial DNA levels were detected in the H9 Dm-dNK expressing cells.     

Synthesis and structural analysis of oxadiazole carboxamide deoxyribonucleoside analogs

Two novel C-linked oxadiazole carboxamide nucleosides 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-5-carboxamide (1) and 5-(2'-deoxy-3',5'-beta-D-erythro-pentofuranosyl)-1,2,4-oxadiazole-3-carboxamide (2) were successfully synthesized and characterized by X-ray crystallography. The crystallographic analysis shows that both unnatural nucleoside analogs 1 and 2 adapt the C2'-endo ("south") conformation. The orientation of the oxadiazole carboxamide nucleobase moiety was determined as anti (conformer A) and high anti (conformer B) in the case of the nucleoside analog 1 whereas the syn conformation is adapted by the unnatural nucleoside 2. Furthermore, nucleoside analogs 1 and 2 were converted with high efficiency to corresponding nucleoside triphosphates through the combination chemo-enzymatic approach. Oxadiazole carboxamide deoxyribonucleoside analogs represent valuable tools to study DNA polymerase recognition, fidelity of nucleotide incorporation, and extension.     

Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase

Purine nucleoside phosphorylase catalyzes reversible phosphorolysis of purine nucleosides and 2'-deoxypurine nucleosides to the free base and ribose (or 2'-deoxyribose) 1-phosphate. Whereas the human enzyme is specific for 6-oxopurine ribonucleosides, the Escherichia coli enzyme accepts additional substrates including 6-oxopurine ribonucleosides, 6-aminopurine ribonucleosides, and to a lesser extent purine arabinosides. These differences have been exploited in a potential suicide gene therapy treatment for solid tumors. In an effort to optimize this suicide gene therapy approach, we have determined the three-dimensional structure of the E. coli enzyme in complex with 10 nucleoside analogs and correlated the structures with kinetic measurements and computer modeling. These studies explain the preference of the enzyme for ribose sugars, show increased flexibility for active site residues Asp204 and Arg24, and suggest that interactions involving the 1- and 6-positions of the purine and the 4'- and 5'-positions of the ribose provide the best opportunities to increase prodrug specificity and enzyme efficiency.     

The N(8)-(2'-deoxyribofuranoside) of 8-aza-7-deazaadenine: a universal nucleoside forming specific hydrogen bonds with the four canonical DNA constituents

The 8-aza-7-deazaadenine (pyrazolo[3,4-d]pyrimidin-4-amine) N(8)-(2'-deoxyribonucleoside) (2) which has an unusual glycosylation position was introduced as a universal nucleoside in oligonucleotide duplexes. These oligonucleotides were prepared by solid-phase synthesis employing phosphoramidite chemistry. Oligonucleotides incorporating the universal nucleoside 2 are capable of forming base pairs with the four normal DNA nucleosides without significant structural discrimination. The thermal stabilities of those duplexes are very similar and are only moderately reduced compared to those with regular Watson-Crick base pairs. The universal nucleoside 2 belongs to a new class of compounds that form bidentate base pairs with all four natural DNA constituents through hydrogen bonding. The base pair motifs follow the Watson-Crick or the Hoogsteen mode. Also an uncommon motif is suggested for the base pair of 2 and dG. All of the new base pairs have a different shape compared to those of the natural DNA but fit well into the DNA duplex as the distance of the anomeric carbons approximates those of the common DNA base pairs.     

Purine Salvage in Two Halophilic Archaea: Characterization of Salvage Pathways and Isolation of Mutants Resistant to Purine Analogs

In exponentially growing cultures of the extreme halophile Halobacterium halobium and the moderate halophile Haloferax volcanii, growth characteristics including intracellular protein levels, RNA content, and nucleotide pool sizes were analyzed. This is the first report on pool sizes of nucleoside triphosphates, NAD, and PRPP (5-phosphoribosyl-α-1-pyrophosphate) in archaea. The presence of a number of salvage and interconversion enzymes was determined by enzymatic assays. The levels varied significantly between the two organisms. The most significant difference was the absence of GMP reductase activity in H. halobium. The metabolism of exogenous purines was investigated in growing cultures. Both purine bases and nucleosides were readily taken up and were incorporated into nucleic acids. Growth of both organisms was affected by a number of inhibitors of nucleotide synthesis. H. volcanii was more sensitive than H. halobium, and purine base analogs were more toxic than nucleoside analogs. Growth of H. volcanii was inhibited by trimethoprim and sulfathiazole, while these compounds had no effect on the growth of H. halobium. Spontaneous mutants resistant to purine analogs were isolated. The most frequent cause of resistance was a defect in purine phosphoribosyltransferase activity coupled with reduced purine uptake. A single phosphoribosyltransferase seemed to convert guanine as well as hypoxanthine to nucleoside monophosphates, and another phosphoribosyltransferase had specificity towards adenine. The differences in the metabolism of purine bases and nucleosides and the sensitivity to purine analogs between the two halobacteria were reflected in differences in purine enzyme levels. Based on our results, we conclude that purine salvage and interconversion pathways differ just as much between the two archaeal species as among archaea, bacteria, and eukarya.     

Ultrastructural localization of plasma membrane-associated urokinase- type plasminogen activator at focal contacts

We have recently shown that urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor type 1 are both found extracellularly beneath cultured human skin fibroblasts and HT-1080 sarcoma cells, but in distinct localizations. Here, the ultrastructural distribution of u-PA was studied using immunoferritin electron microscopy. In HT-1080 cells, u-PA on the extracellular aspect of the plasma membrane was detected at sites of direct contact of the cell with the growth substratum beneath all parts of the ventral cell surface. The ferritin-labeled adhesion plaques, which were enriched in submembraneous microfilaments, were frequently seen at the leading lamellae of the cells as well as in lamellipodia and microspikes. Besides the cell-substratum adhesion plaques, ferritin label was detected at cell-cell contact sites. Double-label immunofluorescence showed a striking colocalization of u-PA and vinculin in both HT-1080 cells and WI-38 lung fibroblasts, which is consistent with u-PA being a focal contact component. The u-PA-containing focal contacts of WI-38 cells had no direct codistribution with fibronectin fibrils. In WI-38 cells made stationary by cultivation in a medium containing 0.5% FCS, vinculin plaques became highly elongated and more centrally located, whereas u-PA immunolabel disappeared from such focal adhesions. These findings show that plasma membrane-associated u-PA is an intrinsic component of focal contacts, where, we propose, it enables directional proteolysis for cell migration and invasion.     

Synthesis,structural studies,and biological evaluation of some purine substituted 1-aminocyclopropane-1-carboxylic acids and 1-amino-1-hydroxymethylcyclopropanes

The novel purine derivatives of 1-aminocyclopropane-1-carboxylic acid (8 and 9) and 1-amino-1-hydroxymethylcyclopropane (12 and 13) with methylene spacer between the base and the cyclopropane ring were prepared by multistep synthetic route involving alkylation of adenine and 6-(N-pyrrolyl)purine with 2-hydroxy-methyl-1-aminocyclopropane-1-carboxylic acid derivative 3 as a key reaction. All novel compounds were racemic. The N-9 substitution of the purine ring and the Z-configuration of the cyclopropane ring in 4-13 were deduced from their 1H and 13C NMR spectra by analyses of chemical shifts, H-H coupling constants and connectivities in two-dimensional homo- and heteronuclear correlation spectra. An unequivocal proof of the stereostructure of 1, 4 and 5 was obtained by their X-ray structure analysis. The novel compounds were evaluated on cytostatic and antiviral activities in several cell lines. The 6-(N-pyrrolyl)purine derivative of 1,2-aminocyclopropane alcohol 12 exhibited a more pronounced inhibitory activity against the proliferation of cervical carcinoma (HeLa) and human fibroblast (WI-38) cells than other types of tumor cell lines. None of the compounds showed inhibitory activities against cytomegalovirus, varicella-zoster virus or other viruses.     

An AZT Analog with Strongly Pairing Ethynylpyridone Nucleobase and Its Antiviral Activity against HSV1

Challenges resulting from novel viruses or new strains of known viruses call for new antiviral agents. Nucleoside analogs that act as inhibitors of viral polymerases are an attractive class of antivirals. For nucleosides containing thymine, base pairing is weak, making it desirable to identify nucleobase analogs that pair more strongly with adenine, in order to compete successfully with the natural substrate. We have recently described a new class of strongly binding thymidine analogs that contain an ethynylmethylpyridone as base and a C-nucleosidic linkage to the deoxyribose. Here we report the synthesis of the 3′-azido-2′,3′-deoxyribose derivative of this compound, dubbed AZW, both as free nucleoside and as ProTide phosphoramidate. As a proof of principle, we studied the activity against Herpes simplex virus type 1 (HSV1). Whereas the ProTide phosphoramidate suffered from low solubility, the free nucleoside showed a stronger inhibitory effect than that of AZT in a plaque reduction assay. This suggests that strongly pairing C-nucleoside analogs of pyrimidines have the potential to become active pharmaceutical ingredients with antiviral activity.