Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.     

Direct PCR amplification of various modified DNAs having amino acids: convenient preparation of DNA libraries with high-potential activities for in vitro selection

We synthesized modified 2'-deoxyuridine triphosphates bearing amino acids at the C5 position and investigated their substrate properties for KOD Dash DNA polymerase during polymerase chain reaction (PCR). PCR using C5-modified dUTP having an amino acyl group (arginyl, histidyl, lysyl, phenylalanyl, tryptophanyl, leucyl, prolyl, glutaminyl, seryl, O-benzyl seryl or threonyl group) gave the corresponding full-length PCR products in good yield. Although dUTP analogues bearing aspartyl, glutamyl or cysteinyl were found to be poor substrates for PCR catalyzed by KOD Dash DNA polymerase, optimization of the reaction conditions resulted in substantial generation of full-length product. In the case of reaction using dUTP analogue having a cysteinyl group, addition of a reducing agent improved the reaction yield. Thus, PCRs using KOD Dash DNA polymerase together with amino acyl dUTP provide convenient and efficient preparation of various modified DNA libraries with potential protein-like activities.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D(2) and D(3) receptors.

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

Reversible stepwise mechanism involving a carbanion intermediate in the elimination of ammonia from L-histidine catalyzed by histidine ammonia-lyase.

L-Histidine labeled with deuterium at the C-5' position of the imidazole ring, L-[5'-2H]histidine (His-5'-D), was used as a probe for investigating a stepwise reversible mechanism via a carbanion intermediate in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine (His-5'-D) (2.45 mM) was incubated with histidine ammonia-lyase (200 units) from Pseudomonas fluorescens at pH 7.0 or 9.0 at 25.0 degrees C for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid in the presence of L-histidine provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The rate of increase in the concentration of urocanic acid exchanged with hydrogen (UA-5'-H) did not depend on the formation rate of urocanic acid and UA-5'-H was continuously formed at a constant rate (25.6 microM/h) even after the completion of urocanic acid formation. These observations suggested the presence of the reversible reaction of urocanic acid and a carbanion intermediate. Since there was only a minor contribution for the formation of UA-5'-H from L-histidine exchanged with solvent hydrogen (His-5'-H), the main pathway in the enzymatic reaction of His-5'-D must be the formation of UA-5'-D via a carbanion intermediate (carbanion-D). Regeneration of the carbanion-D from UA-5'-D by its reverse reaction and subsequent hydrogen incorporation at C-5' would contribute to a large extent for the formation of UA-5'-H. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.     

Synthesis of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole analogues and their binding affinities for dopamine D2 and D3 receptors

A series of 2-(2,3-dimethoxyphenyl)-4-(aminomethyl)imidazole derivatives was prepared and their affinity for dopamine D₂ and D₃ receptors was measured using in vitro binding assays. Several oxadiazole analogues were also prepared and tested for their affinity for dopamine D₂ and D₃ receptors. The results of receptor binding studies indicated that the incorporation of an imidazole moiety between the phenyl ring and the basic nitrogen did not significantly increase the selectivity for dopamine D₃ receptors, whereas the incorporation of an oxadiazole at the same region resulted in a total loss of affinity for both dopamine receptor subtype binding sites. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-4-(6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolinomethyl)imidazole (5i), which has a D₃ receptor affinity of 21 nM and a 7-fold selectivity for D₃ versus D₂ receptors. The binding affinity for σ₁ and σ₂ receptors was also measured, and the results showed that several analogues were selective σ₁ receptor ligands.     

Substrate properties of C5-substituted pyrimidine 2'-deoxynucleoside 5'-triphosphates for thermostable DNA polymerases during PCR

In order to enhance a collection of modified deoxynucleoside triphosphates useful for in vitro selection or SELEX (systematic evolution of ligands by exponential enrichment) techniques, we designed and synthesized modified analogues of 2'-deoxyuridine triphosphate and 2'-deoxycytidine triphosphate bearing a flexible and hydrophilic 7-amino-2,5-dioxaheptyl linker at a C5 position. Both analogues were found to be substrates for thermostable DNA polymerases which belong to an evolutional family B during PCR.     

Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1).

Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-2. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the epsilon-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.     

High transfection efficiency of poly(4-vinylimidazole) as a new gene carrier

Poly(4-vinylimidazole) (P4V) was obtained by free radical polymerization of 4-vinylimidazole (4V) prepared by decarboxylation of urocanic acid. P4V formed a complex with DNA that exhibited higher transfection effiency on Hela cells than polyethylenimine (PEI), through the proton sponge mechanism of the imidazole groups in the side chain of the P4V, and low cell toxicity.     

Quantitation of fluorescent nucleotide incorporation by capillary gel electrophoresis and laser-induced fluorescence detection

A method has been developed by which enzymatically incorporated fluorophore-labeled nucleotide sites in nucleic acid can be quantitated by degradation of nanogram quantities of DNA followed by capillary gel electrophoretic analysis with fluorescence detection. In this way the differing relative labeling densities achieved using either C5-substituted dUTP's or N4-substituted dCTP's were determined. The method has proven to be very useful in obtaining quantitative analytical data from the small quantities of complex molecules produced in nick translations. Various polymerization conditions using DNA polymerase I were examined to determine optimal labeling density. Simultaneous copolymerization of green fluorescing dCTP and dUTP nucleotides were undertaken in an attempt to maximize labeling density.     

Inhibition of DNA polymerase reactions by pyrimidine nucleotide analogues lacking the 2-keto group.

To investigate the influence of the pyrimidine 2-keto group on selection of nucleotides for incorporation into DNA by polymerases, we have prepared two C nucleoside triphosphates that are analogues of dCTP and dTTP, namely 2-amino-5-(2'-deoxy-beta-d-ribofuranosyl)pyridine-5'-triphosphate (d*CTP) and 5-(2'-deoxy- beta-d-ribofuranosyl)-3-methyl-2-pyridone-5'-triphosphate (d*TTP) respectively. Both proved strongly inhibitory to PCR catalysed by Taq polymerase; d*TTP rather more so than d*CTP. In primer extension experiments conducted with either Taq polymerase or the Klenow fragment of Escherichia coli DNA polymerase I, both nucleotides failed to substitute for their natural pyrimidine counterparts. Neither derivative was incorporated as a chain terminator. Their capacity to inhibit DNA polymerase activity may well result from incompatibility with the correctly folded form of the polymerase enzyme needed to stabilize the transition state and catalyse phosphodiester bond formation.     

Impermeant auxin analogues have auxin activity

Protein conjugates of 5-aminonaphthalene-1-acetic acid and of 5-azido-naphthalene-1-acetic acid have been prepared and evaluated for auxin activity in two types of assay. In standard elongation tests with pea (Pisum sativum L.) epicotyl sections the conjugates are inactive. However, if the epicotyls are abraded to perforate the cuticle, auxin activity is observed provided that the conjugates are not too large to traverse the cell wall. In a system lacking a cell wall — tobacco (Nicotiana tabacum L.) protoplasts — conjugates of widely differing size are able to induce membrane hyperpolarization. These results support other recent evidence that auxin receptors are exposed at the exterior face of the plasma membrane and indicate that auxins can produce both rapid and longer-term responses without entering the cell.Abbreviations ABP auxin-binding protein - BSA bovine serum albumin - E_m transmembrane potential difference - KLH keyhole limpet hemocyanin - NAA naphthalene-1-acetic acid This work was partly supported under the Biotechnology Action Programme of the European Economic Communities. We thank Mr. P. Cozens for technical assistance.To whom correspondence should be addressed.     

Studies on antibiotic biosynthesis by protoplasts and resting cells of Streptomyces echinatus. Part II. Effect of chromophore precursors

Washed cell and protoplast suspensions from Streptomyces echinatus A8331, which produces the quinoxaline antibiotic echinomycin, have been used to study the effects of analogues of the natural chromophore upon antibiotic biosynthesis. Addition of quinoline-2-carboxylic acid caused a decrease in the labelling of echinomycin from L-[methyl-14C]methionine and an increase in labelled chloroform-extractable material. Quinoxaline-2-carboxylic acid increased the incorporation of radioactivity into both fractions. Thieno[3,2-b]pyridine-5-carboxylic acid, 6-methylquinoline-2-carboxylic acid, and quinoline-2-carboxylic acid (also to a lesser extent 7-chloroquinoxaline-2-carboxylic acid) increased markedly the incorporation of radioactivity into chloroform-extractable material and virtually abolished echinomycin synthesis. Autoradiographs of extracts from suspensions supplemented with the latter four analogues revealed bis-substituted metabolites not found in unsupplemented cultures. When protoplast suspensions were incubated with L-[U-14C]serine, L-[U-14C]valine, or DL-[benzene ring-U-14C]tryptophan, quinoline-2-carboxylic acid, thieno[3,2-b]pyridine-5-carboxylic acid, and 6-methylquinoline-2-carboxylic acid directed the synthesis of antibiotically active bis derivatives at the expense of echinomycin. When analogues of quinoxaline-2-carboxylic acid previously found unsuitable for incorporation by growing cultures were tested in protoplast suspensions, only isoquinoline-3-carboxylic acid caused a large increase in the incorporation of radioactivity from L-[methyl-14C]methionine into chloroform-extractable material. With DL-[benzene ring-U-14C]tryptophan as the radiolabel, benzotriazoline-2-acetic acid and 6-bromoquinoxaline-2-carboxylic acid as well as isoquinoline-3-carboxylic acid sharply reduced the labelling of echinomycin.     

Effects of nucleotide analogues on Euplotes aediculatus telomerase processivity: evidence for product-assisted translocation.

Telomerase is a unique ribonucleoprotein that reverse transcribes a defined region of its RNA subunit onto the ends of eukaryotic chromosomes. The product of telomerase, telomeric DNA, is typically a G-rich repeated sequence, (TTTTGGGG)(n) in the ciliate Euplotes aediculatus and (TTAGGG)(n) in humans. Telomerase can extend oligonucleotide primers in vitro in a processive fashion. We used dNTP analogues to study the structure-activity relationship between substrate nucleotides and processivity of telomerase from E. aediculatus. Several analogues, including 2'-deoxyuridine triphosphate (dUTP), 2'-deoxyinosine triphosphate (dITP), and 7-deaza-2'-deoxyguanosine triphosphate (7-deaza-dGTP), were good substrates for telomerase with K(m) and V(max) values near those of the natural substrates, dTTP and dGTP. However, telomerase processivity was affected with these substrates, decreasing in the order dUTP > 7-deaza-dGTP > dITP. Telomerase did not completely reverse transcribe the template when dITP was the substrate, and it efficiently extended a primer by the addition of two repeats when 7-deaza-dGTP and dUTP were utilized. When the same nucleotide analogues were incorporated into the primers, no effects were observed except in the case of a 3'-terminal deoxyinosine. The data support a model that includes the formation of an intramolecular secondary structure within the product DNA to facilitate translocation. The most likely structure is a G-G hairpin.     

Differential inhibitory effects of 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates and related nucleotides on DNA-dependent RNA polymerases I and II from the cherry salmon (Oncorhynchus masou)

Various 5-substituted 1-beta-D-xylofuranosyluracil 5'-triphosphates (hydrogen, methyl-, ethyl-, n-propyl, n-butyl, fluoro-, chloro-, bromo-, and iodo derivatives) and some of the 3'-deoxyribofuranosyl nucleotides (3'-deoxy UTP and 3'-deoxy TTP) were synthesized chemically and their inhibitory effects on DNA-dependent RNA polymerases I and II of the cherry salmon (Oncorhynchus masou) were studied systematically. These 3'-modified UTP analogues could not be utilized as substrates in place of UTP, but they did inhibit the incorporation of UMP into RNA in vitro. In contrast, 2'-modified UTP analogues, such as 2'-dTTP and Ara TTP, were neither substrates nor inhibitors. Kinetic analysis showed that the inhibition by these compounds was essentially competitive with substrate UTP. The K1 values of RNA polymerase I for the analogues were smaller (2-6 microM) than the Km value for UTP (8 microM), but those for xylo-EtUTP, xylo-PrUTP, and xylo-BuUTP were larger (about 20 microM) than the Km for UTP. In contrast to these alkyl groups with steric and electron-donating effects, halogen groups have electron-withdrawing effects on the uracil nucleus. Therefore, it was concluded that the inhibitory activity of these analogues on RNA polymerase I was not affected by the inductive effects of substituent groups at the 5-position of uracil nucleus but by their steric effects. On the other hand, all of the K1 values of RNA polymerase II for UTP analogues were smaller (0.4-3 microM) than the Km value for UTP (4 microM). In this case, neither steric effect nor an inductive effect of substituents on UTP analogues influenced the inhibitory activity towards RNA polymerase II.     

(2-Aryl-5-methylimidazol-4-ylcarbonyl)guanidines and (2-aryl-5-methyloxazol-4-ylcarbonyl)guanidines as NHE-1 inhibitors

A series of (2-aryl-5-methylimidazol-4-ylcarbonyl)guanidines and (2-aryl-5-methyloxazol-4-ylcarbonyl)guanidines were synthesized and evaluated as NHE-1 inhibitors. The structure–activity relationships well matched those of furan derivatives, which were previously investigated. The (2,5-disubstituted)phenyl compounds showed better activities than the other analogues in both imidazole and oxazole compounds. Especially, 2-(2,5-dichlorophenyl)imidazole 52, and 2-(2-methoxy-5-chlorophenyl)imidazole 54 compounds exhibited potent cardioprotective efficacy both in vitro and in vivo as well as high NHE-1 inhibitory activities.     

The Efficiency of DNA Labeling with Near-Infrared Fluorescent Dyes

The efficiency of DNA labeling was assessed for 2'-deoxyuridine 5'-triphosphate (dUTP) derivatives containing the Cy7 cyanine dye as a fluorophore. Two fluorescent Cy7-labeled dUTP analogs differed in the chemical structure of the linker between the fluorophore and nucleotide moieties. The efficiency of the polymerase chain reaction (PCR) and inhibition with modified nucleotides were estimated by real-time PCR. The efficiency of labeled nucleotide incorporation in PCR products was measured by quantitative electrophoresis. The efficiency of target DNA labeling was evaluated by binding the fluorescently labeled PCR products to a microarray of oligonucleotide probes immobilized in hydrogel drops (a biochip). The near-infrared hybridization signal was detected by digital luminescence microscopy. An increase in linker length was found to provide more efficient incorporation of the labeled nucleotide. Both of the compounds provided high sensitivity and high specificity of DNA testing via allele-specific hybridization on a biochip.

     

Effect of the carcinogenic metabolites of aromatic amino acids on oxidative processes in lipids in vitro and in vivo

A comparative study of the effect produced by endogenous carcinogens (p-hydroxyphenyllactic and 5-methoxyindole-3-acetic acids) and their non-cancerogenic analogues on lipid peroxidation in vitro and in vivo was performed. It has been found that cancerogenic tyrosine and serotonin metabolites, unlike their non-cancerogenic analogues, increase lipid peroxidation in vitro. In vivo, cancerogenic tyrosine metabolite--p-hydroxyphenyllactic acid--is capable both of increasing and decreasing antioxidative lipid activity in the animal liver.     

Evaluation of analogues of GalNAc as substrates for enzymes of the mammalian GalNAc salvage pathway

Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-Acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans, and glycoproteins; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc, which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogues of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, galactokinase 2 (GK2) and uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogues that can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway: a modification on C6 is not tolerated by GK2; AGX1 can use all products of GK2 although with various efficiencies; and all analogues transformed into UDP-GalNAc analogues except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogues that could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detection on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogues can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism, and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.