Buffer catalyzed cleavage of uridylyl-3',5'-uridine in aqueous DMSO: comparison to its activated analog, 2-hydroxypropyl 4-nitrophenyl phosphate.

Buffer catalysis of the cleavage and isomerization of uridylyl-3',5'-uridine (UpU) has been studied over a wide pH range in 80% aq. DMSO. The diminished hydroxide ion concentration in this solvent system made catalysis by amine buffers (morpholine, 4-hydroxypiperidine and piperidine) visible even at relatively low buffer concentrations (10-200 mmol L(-1)). The observed catalysis was, however, much weaker than what has been previously reported for the activated RNA model 2-hydroxypropyl 4-nitrophenyl phosphate (HPNP) in the same solvent system. In the case of morpholine, contribution of both the acidic and the basic buffer constituent was significant, whereas with 4-hydroxypiperidine and piperidine participation of the acidic constituent could not be established unambiguously. The results underline the importance of using realistic model compounds, along with activated ones, in the study of the general acid/base catalysis of RNA cleavage.

Hydrolysis of dinucleoside phosphates - mRNA 5' cap analogues - promoted by a binuclear copper(II)-zinc(II) complex.

The hydrolysis of a 5' cap analogue, diadenosinyl-5',5'-triphosphate (ApppA), and two dinucleoside monophosphates: adenylyl(3',5')adenosine (ApA) and uridylyl(3',5')uridine (UpU) promoted by an imidazolate-bridged heterobinuclear copper(II)-zinc(II) complex, Cu(II)-diethylenetriamino-micro-imidazolato-Zn(II)- tris(aminoethyl)amine trisperchlorate (denoted as Cu,Zn-complex in the followings) has been investigated. Kinetic measurements were performed in order to explore the effects of pH, the total concentration of the Cu,Zn-complex and temperature on the cleavage rate. The catalytic activity of the Cu,Zn-complex was quantified by pseudo-first-order rate constants obtained in the excess of the cleaving agent. The results show that the Cu,Zn-complex and its deprotonated forms have phosphoesterase activity and with ApppA the metal complex promoted cleavage takes place selectively within the triphosphate bridge.

Condensation of triformylmethane with guanosine.

Guanosine has been reacted with triformylmethane (TFM) in refluxing pyridine. Four different products, 4-7, were isolated by preparative RP-HPLC, and characterized by 1H and 13C NMR and UV spectroscopy and mass spectrometry. One of the products. the cyclic 1:1 adduct 4, is a stable cyclic carbinolamine formed probably by cyclization of the expected aminomethylene derivative 3. Compound 4 then undergoes reversible dehydration to the fully conjugated adduct 5. The appearance of the additional adducts, 6 and 7, suggests that TFM is prone to transformations resulting in the formation of methylenemalonaldehyde (9) and 1,1,3,3-tetraformylpropane (11).

The effect of secondary structure on cleavage of the phosphodiester bonds of RNA.

This review discusses the effects the secondary structure of an RNA molecule has on the inherent reactivity of its phosphodiester bonds, and on the catalytic activity of metal ion-based cleaving agents. The basic principles of the intramolecular transesterification of RNA phosphodiester bonds, particularly cleavage, are first briefly described. Studies of the structural effects on the cleavage, in the absence and in the presence of metal ion catalysts, are then reviewed, and the sources of the reactivity differences observed in different structures are discussed.

The chemical stability of S-(2-acylthioethyl) and S-acyloxymethyl protected thymidylyl-3',5'-thymidine phosphoromonothiolates and their deacylation products in aqueous solution.

The hydrolytic stability of the S-(2-acetylthioethyl) (1a,b), S-(2-pivaloylthioethyl) (2a,b), and S-acetyloxymethyl (3a,b) protected Rp and Sp phosphoromonothiolates of 3',5'-TpT has been studied. Rather unexpectedly, an intramolecular hydroxide ion catalyzed acetyl migration from the protecting group to the nucleoside 3'- and 5'-hydroxy functions was found to compete with the intermolecular displacement of the AcSCH2CH2S- or AcOCH2S-ligand from the phosphorus atom of 1a,b and 3a,b, respectively. With the S-pivaloylthioethyl derivative 2a,b no such reaction took place. Additionally, the kinetics of the cleavage of the S-(2-mercaptoethyl) group from 4a,b, the products of enzymatic deacylation of 1a,b and 2a,b, were studied as a function of pH.

Preparation, hydrolysis and intramolecular transesterification of 3'-deoxy-3'-thioinosine 3'-S-dimethylphosphorothiolate.

The hydrolytic reactions of the dimethyl ester of 3'-deoxy-3'-thioinosine 3'-S-phosphorothiolate have been followed over a wide aciditiy range by HPLC. At pH > 3, only hydroxide ion catalyzed isomerization to the 2'-dimethylphosphate takes place, whereas under more acidic conditions hydrolysis to the 2'-monomethylphosphate and 3'-S-monomethylphosphorothiolate competes. The latter is the only product accumulating in very acidic solutions (1 M hydrochloric acid). Mechanisms of the reactions are discussed.

Hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine: kinetics and mechanisms for the cleavage, desulfurization, and isomerization of the internucleosidic linkage

The hydrolytic reactions of the phosphorodithioate analogue of uridylyl(3',5')uridine [3',5'-Up(s)2U] were followed by HPLC over a wide pH range at 363.2 K. Under acidic and neutral conditions, three reactions compete: (i) desulfurization to a mixture of the (Rp)- and (Sp)-diastereomers of the corresponding 3',5'- and 2',5'-phosphoromonothioates [3',5'- and 2',5'-Up(s)U], which are subsequently desulfurized to a mixture of uridylyl(3',5')- and -(2',5')uridine [3',5'- and 2',5'-UpU], (ii) isomerization to 2',5'-Up(s)2U, and (iii) cleavage to uridine, in all likelihood via a 2',3'-cyclic phosphorodithioate (2',3'-cUMPS2). Under alkaline conditions (pH > 8), only a hydroxide ion catalyzed hydrolysis to uridine via 2',3'-cUMPS2 takes place. At pH 3-7, all three reactions are pH-independent, the desulfurization being approximately 1 order of magnitude faster than the cleavage and isomerization. At pH < 3, all the reactions are hydronium ion catalyzed. On going to very acidic solutions, the cleavage gradually takes over the desulfurization and isomerization. Accordingly, the cleavage overwhelmingly predominates at pH < 0. The overall hydrolytic stability of 3',5'-Up(s)2U is comparable to that of (Sp)- and (Rp)-3',5'-Up(s)U (and to that of 3',5'-UpU, except at pH < 2). The rate of the hydroxide ion catalyzed hydrolysis of 3',5'-Up(s)2U is 37% and 53% of that of (Sp)- and (Rp)-3',5'-Up(s)U, respectively. The reactions, however, differ with the respect of the product accumulation. While the phosphoromonothioates produce a mixture of 2'- and 3'-thiophosphates as stable products, 3',5'-Up(s)2U is hydrolyzed to uridine without accumulation of the corresponding dithiophosphates. At pH < 3, where the hydrolysis is hydronium ion catalyzed, the kinetic thio-effect of the second thio substitution is small: under very acidic conditions (Ho -0.69), (Sp)-3',5'-Up(s)U reacts 1.6 times as fast as 3',5'-Up(s)2U, but the reactivity difference decreases on going to less acidic solutions. In summary, the hydrolytic stability of 3',5'-Up(s)2U closely resembles that of the corresponding phosphoromonothioate. While replacing one of the nonbridging phosphate oxygens of 3',5'-UpU with sulfur stabilizes the phosphodiester bond under acidic conditions by more than 1 order of magnitude, the replacement of the remaining nonbridging oxygen has only a minor influence on the overall hydrolytic stability.

Sulfuric acid-catalyzed acetolysis of anomeric methyl 2,3,4,6-tetra-O-acetyl-D-mannopyranosides: kinetics and mechanism.

The kinetics of the acetolysis and accompanying anomerization of methyl 2,3,4,6-tetra-O-acetyl-alpha- and -beta-D-mannopyranosides at different concentrations of sulfuric acid in acetic anhydride-acetic acid mixtures were studied. The progress of the reactions was followed by gas chromatography, and the rate constants of the partial reactions were calculated on the basis of the time-dependent product distribution obtained. The mechanisms of the reactions involved are discussed. The involvement of unstable ionic intermediates is taken into account in the evaluation of the kinetic results, and simplified and extended models are used in the mathematical treatment of the results. A fourth-order Runge-Kutta algorithm is used to calculate rate constants. Acetolysis was found to be faster for mannosides than for glucosides relative to their anomerization. The beta-mannopyranoside prefers endocyclic CO-bond rupture, while in the alpha anomer the endocyclic and exocyclic cleavages are comparatively rapid.

Cleavage of the phosphodiester bond of uridylyl-(3',5')-8-carboxymethylaminoadenosine by hydronium, hydroxide and zinc(II) ions: a model study aimed at elucidating the potential of a carboxylate function as an intramolecular catalyst.

Uridylyl-(3',5')-8-carboxymethylaminoadenosine has been synthesised, and its transesterification to uridine 2',3'-cyclic phosphate in the presence and absence of Zn2+ ion has been studied. The results show that a carboxylate function in the vicinity of the phosphodiester bond accelerates the metal ion promoted cleavage but not the metal ion independent reaction. Under acidic conditions, the predominant reaction is the cleavage of the side chain, giving the 8-amino derivative.

Aminooxy functionalized oligonucleotides: preparation, on-support derivatization, and postsynthetic attachment to polymer support.

Three novel phosphoramidites, each bearing a phthaloyl-protected aminooxy tail, were prepared and applied in automated oligonucleotide synthesis. After chain assembly, the phthaloyl protection was removed with hydrazinium acetate. Normal succinyl linker turned to be stable under these conditions, and hence the support-bound oligonucleotide could be converted to a pyrene oxime conjugates by reacting with pyrene carbaldehyde or cis-retinal. Standard ammonolytic deprotection then released the deprotected conjugate in solution. Alternatively, the crude aminooxy-tethered oligonucleotide was immobilized to microscopic polymer particles by reacting the aminooxy function with the particle-bound aldehyde or epoxide groups. These immobilized oligonuceotides were shown to serve properly as probes in a mixed phase hybridization assay.

Peptide-oligonucleotide phosphorothioate conjugates with membrane translocation and nuclear localization properties.

Eighteen peptide-oligonucleotide phosphorothioate conjugates were prepared in good yield and thoroughly characterized with electrospray ionization mass spectra. When applied to the living cells, conjugates exhibiting membrane translocation and nuclear localization properties displayed efficient intracellular penetration but failed to show any serious antisense effect. Studies on the intracellular distribution of the fluorescein-labeled conjugates revealed their trapping in endosomes.

Mass spectrometric (HPLC/ESI--MS/MS) quantification of pyrimido[1,3-a]purin-10(3H)-one, a guanine adduct formed by reaction of malondialdehyde with DNA.

A high performance liquid chromatography/electrospray ionization tandem mass spectrometric (HPLC/ESI MS/MS) method has been developed for quantification of pyrimido[1,2-a]purin-10(3H)-one adducts from DNA. The method is based on acid-catalyzed cleavage of the adducts from DNA and the use of [2,3a,10-13C3]pyrimido[1,2-a]purin-10(3H)-one as an internal standard in the analysis. For this purpose the latter compound was prepared. Rate constants for the acid-catalyzed cleavage of pyrimido[1,2-a]purin-10(3H)-one from the corresponding 2'-deoxyribonucleoside were determined, and its hydrolytic stability and possible formation by a cross reaction between guanine and [2,3a,10]pyrimido[1,2-a]purin-10(3H)-one were studied.

Simultaneous detection of several oligonucleotides by time-resolved fluorometry: the use of a mixture of categorized microparticles in a sandwich type mixed-phase hybridization assay.

Porous, uniformly sized (50 micrometer) glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as a solid phase to construct a sandwich type hybridization assay that allowed simultaneous detection of up to six oligonucleotides from a single sample. The assay was based on categorization of the particles by two organic prompt fluorophores, viz. fluorescein and dansyl, and quantification of the oligonucleotide hybridization by time-resolved fluorometry. Accordingly, allele-specific oligodeoxyribonucleotide probes were assembled on the particles by conventional phosphoramidite strategy using a non-cleavable linker, and the category defining fluorescein and/or dansyl tagged building blocks were inserted in the 3'-terminal sequence. An oligonucleotide bearing a photoluminescent europium(III) chelate was hybridized to the complementary 3'-terminal sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound allele-specific probes via the 5'-terminal sequence of the target. After hybridization each individual particle was subjected to three different fluorescence intensity measurements. The intensity of the prompt fluorescence signals of fluorescein and dansyl defined the particle category, while the europium(III) chelate emission quantified the hybridization. The length of the complementary region between the target oligonucleotide and the particle-bound probe was optimized to achieve maximal selectivity. Furthermore, the kinetics of hybridization and the effect of the concentration of the target oligomer on the efficiency of hybridization were evaluated. By this approach the possible presence of a three base deletion (DeltaF508), point mutation (G542X) and point deletion (1078delT) related to cystic fibrosis could unequivocally be detected from a single sample.

Metal ion-dependent hydrolysis of RNA phosphodiester bonds within hairpin loops. A comparative kinetic study on chimeric ribo/2'-O-methylribo oligonucleotides.

Several chimeric ribo/2'- O -methylribo oligonucleotides were synthesized and their hydrolytic cleavage studied in the presence of Mg2+, Zn2+, Pb2+and the 1,4,9-triaza-cyclododecane chelate of Zn2+(Zn2+[12]aneN3) to evaluate the importance of RNA secondary structure as a factor determining the reactivity of phosphodiester bonds. In all the cases studied, a phosphodiester bond within a 4-7 nt loop was hydrolytically more stable than a similar bond within a linear single strand, but markedly less stable than that in a double helix. With Zn2+and Zn2+[12]aneN3, the hydrolytic stability of a phosphodiester bond within a hairpin loop gradually decreased on increasing the distance from the stem. A similar but less systematic trend was observed with Pb2+. Zn2+- and Pb2+-promoted cleavage was observed to be considerably more sensitive to the secondary structure of the chain than that induced by Zn2+[12]aneN3. This difference in behaviour may be attributed to bidentate binding of uncomplexed aquo ions to two different phosphodiester bonds. Mg2+was observed to be catalytically virtually inactive compared with the other cleaving agents studied.

A fluorescence spectroscopic study on the binding of mRNA 5'-cap-analogs to human translation initiation factor eIF4E: a critical evaluation of the sources of error.

Equilibrium constants for the association of human protein translation initiation factor eIF4E with two mRNA 5'-cap analogs, namely 7-methylguanosine 5'-triphosphate and P1-(7-methylguanosine-5') P3-(guanosine-5') triphosphate, and with guanosine 5'-monophosphate have been redetermined by the fluorescence quenching method taking the inner filter effect of the cap-analog into account. It has been shown that neglecting the latter correction may lead to either underestimation or overestimation of the association constant obtained by applying the Eadie-Hofstee plot: the reasonably firm binding of 7-methylated cap-analogs becomes underestimated, while the weak binding of non-methylated nucleotides becomes overestimated.

Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: quantitation and optimization of a sandwich type assay.

Uniformly sized (50 micro m) porous glycidyl methacrylate/ethylene dimethacrylate particles (SINTEF) were used as the solid phase in a sandwich type mixed-phase hybridization assay based on time-resolved fluorescence detection on a single particle. These particles were coated with oligodeoxyribonucleotide probes by conventional phosphoramidite chain assembly. An oligodeoxyribonucleotide bearing a photoluminescent europium(III) chelate, ¿2,2',2",2"'-¿¿4'-¿4"'-[(4, 6-dichloro-1,3,5-triazin-2-yl)amino]phenyl¿-2,2':6',2"-terpyrid ine-6, 6"-diyl¿bis(methylenenitrilo)¿tetrakis(acetato)¿eur opi um(III), was hybridized to a complementary sequence of the target oligonucleotide, and the resulting duplex was further hybridized to the particle-bound probes. The latter binding was quantified by time-resolved measurement of the emission signal of a single particle. Kinetics of hybridization and the effect of the concentration of the target oligomer and the fluorescently tagged probe on the efficiency of hybridization were studied. The intensity of the emission signal was linearly related to the concentration of the target oligomer over a range of 5 orders of magnitude. The length of the complementary region between the target oligomer and the particle-bound probe was varied, and the effect of point mutations and deletions on the hybridization efficiency was determined in each case. The maximal selectivity was observed with 10-16-base pair complementary sequences, the optimal length depending on the oligonucleotide loading on the particle. Discrimination between the complete matches and point mismatches was unequivocal, a single point mutation and/or deletion decreasing the efficiency of hybridization by more than 2 orders of magnitude.

Disulfide-tethered solid supports for synthesis of photoluminescent oligonucleotide conjugates: hydrolytic stability and labeling on the support.

Several new disulfide-tethered solid supports (S1-S5) were synthesized, and their resistance against ammonolysis was tested. Among these supports, only the one bearing an N-[15-[(4, 4'-dimethoxytrityl)oxy]-12,13-dithiapentadecanoyl] linker (S4b) tolerated ammonolysis and exhibited properties compatible with the oligonucleotide synthesis by phosphoramidite strategy. The applicability of this disulfide linker structure in postsynthetic oligonucleotide labeling on the support was demonstrated by introduction of two photoluminescent lanthanide chelates or two dansyl groups to the N4-(6-aminohexyl) amino-modified cytosine residues at the 5' end of the oligonucleotide sequence. Subsequent release of the resulting conjugates as their 3'-phosphates was achieved by reductive cleavage of the disulfide bond and precipitation of the conjugate from the solution with ethanol. The fluorescently tagged oligomer obtained showed hybridization properties similar to those of oligonucleotides labeled in solution.

A series of 6-(omega-methanesulfonylthioalkoxy)-2-N-methyl- 1,2,3, 4-tetrahydroisoquinolines: cysteine-reactive molecular yardsticks for probing alpha2-adrenergic receptors.

A series of 6-(omega-methanesulfonylthioalkoxy)-2-N-methyl-1,2,3, 4-tetrahydroisoquinolines (7a-d) was prepared and characterized as SH-reactive molecular yardsticks useful in probing alpha2-adrenergic receptors. Rapid displacement of the methanesulfonyl group by a cysteine residue in dilute aqueous solution with concomitant formation of a disulfide conjugate was verified by MALDI-TOF mass spectrometric analysis of the reaction of 7a with a cysteine-containing decapeptide. 7a-d all showed a marked affinity for the three different variants of human alpha2-adrenergic receptors: H alpha(2A)wt, H alpha(2B)wt, and mutant H alpha(2A)Ser201Cys197. However, only the mutated receptor (H alpha(2A)Ser201Cys197) was irreversibly inactivated, and the extent of inactivation in this case was linearly dependent on the length of the side chain of 7a-d. These results show that the molecular yardstick approach tested here can provide useful information for modeling receptor proteins.

Sensitive bioaffinity assays with individual microparticles and time-resolved fluorometry.

Future immunoassays and nucleic acid hybridization assays will be performed in miniaturized formats that utilize microchips or microparticles. This will require a sensitive detection technology that allows spatial resolution. By using fluorescent europium chelates and time-resolved microfluorometry, one can detect 11,000 europium molecules on individual microparticles. In a miniaturized noncompetitive immunoassay of prostate-specific antigen (PSA), we quantitatively detected 5 ng/L (0.05 amol per particle) of the analyte on an individual microparticle with excellent precision over the whole measurement range (CV <10%). Using a hybridization assay, we also could detect the deltaF508 mutation for cystic fibrosis on individual microparticles. Consequently, fluorescent lanthanide chelate labels and time-resolved microfluorometry qualify as the next generation of technology in this field.

Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.

Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.