Nonbridging Position Research Articles

The role of the Hint1 protein in the metabolism of phosphorothioate oligonucleotides drugs and prodrugs, and the release of H2S under cellular conditions

Phosphorothioate oligonucleotides (PS-oligos) containing sulfur atom attached in a nonbridging position to the phosphorus atom at one or more internucleotide bond(s) are often used in medicinal applications. Their hydrolysis in cellular media proceeds mainly from the 3′-end, resulting in the appearance of nucleoside 5′-O-phosphorothioates ((d)NMPS), whose further metabolism is poorly understood. We hypothesize that the enzyme responsible for (d)NMPS catabolism could be Hint1, an enzyme that belongs to the histidine triad (HIT) superfamily and is present in all organisms. We previously found that (d)NMPS were desulfurated in vitro to yield (d)NMP and H2S in a Hint1-assisted reaction. Here, we demonstrate that AMPS/GMPS/dGMPS introduced into HeLa/A549 cells are intracellularly converted into AMP/GMP/dGMP and H2S. The level of the released H2S was relative to the concentration of the compounds used and the reaction time. Using RNAi technology, we have shown decreased levels of AMPS/GMPS desulfuration in HeLa/A549 cells with reduced Hint1 levels. Finally, after transfection of a short Rp-d(APSAPSA) oligomer into HeLa cells, the release of H2S was observed. These results suggest that the metabolic pathway of PS-oligos includes hydrolysis into (d)NMPS (by cellular nucleases) followed by Hint1-promoted conversion of the resulting (d)NMPS into (d)NMP accompanied by H2S elimination. Our observations may be also important for possible medicinal applications of (d)NMPS because H2S is a gasotransmitter involved in many physiological and pathological processes.

ChemInform Abstract: Development of Metal Ion Containing Catalysts for the Decomposition of Phosphorothioate Esters

AbstractReview: 75 refs.

Development of metal-ion containing catalysts for the decomposition of phosphorothioate esters

The widespread use of phosphorothioate esters as agricultural pesticides, chemical weapons and mechanistic probes in enzymology has sparked interest in the reactivity of these thio-substituted analogues of phosphate esters. In this brief account, we summarize the recent developments in our understanding of the mechanisms of hydrolysis (and solvolysis in methanol) of phosphorothioates containing a sulfur atom in the bridging and/or non-bridging position. A small number of highly efficient catalytic systems containing the metal ions La(III), Pd(II), Cu(II) and Zn(II) have been developed to promote the degradation of the various classes of phosphorothioate esters. The mechanisms of the base promoted solvolytic reactions in water and methanol and those of the metal catalyzed cleavage are presented, as well as a discussion of the energetics of the catalytic processes and other salient features. The aim of this review is to provide the reader with a contemporary physical organic description of phosphorothioate ester cleavage. This article is part of a Special Issue entitled: Chemistry and mechanism of phosphatases, diesterases and triesterases.

Unique Catalytic Mechanism of Phosphatidylinositol-Specific Phospholipase C fromStreptomyces antibioticus

Calcium-dependent phosphatidylinositol-specific phospholipase C from Streptomyces antibioticus (saPLC1) catalyzes hydrolysis of phosphatidylinositol (PI) into inositol 1-phosphate by a unique mechanism involving formation of inositol 1,6-cyclic phosphate (1,6-IcP) as an intermediate. This work examines the rates and products of cleavage of phosphorothioate and phosphorodithioate analogues of PI in which sulfur was introduced into the phosphate moiety at a nonbridging position (pro-R or pro-S), a bridging position, or both. The replacement of the pro-S oxygen in the phosphoryl moiety of PI by sulfur results in a 3 x 10(7)-fold decrease of the catalytic rate constant, whereas alteration of the pro-R oxygen results in only a modest rate reduction. The addition of the second sulfur atom into the bridging position of the S(p) isomer of the phosphorothioate analogue causes a dramatic (2 x 10(5)-fold) increase of the rate of cleavage but has a negligible effect on the R(p) isomer. These differences are consistent with a change in the mechanism for the S(p) isomer of the phosphorodithioate analogue into a more dissociative type, where the leaving group carries a large amount of negative charge. In addition, hydrolysis of the diastereomers of the phosphorothioate analogues of 1,6-IcP, inositol cis-1,6-IcPs and inositol trans-1,6-IcPs, affords two distinct products, inositol 1-phosphorothioate and inositol 6-phosphorothioate, respectively. Formation of inositol 6-phosphorothioate is explained by the binding of trans-1,6-IcPs in the active site in a rotated orientation that interchanges the oxygen atoms at the 1- and 6-positions, thereby allowing the hydroxyl group at the 1-position to act as a leaving group. The reorientation of the intermediate is driven by formation of favorable interactions of the enzyme active site with the nonbridging oxygen in the trans intermediate.

Mechanism of Dimethylallyltryptophan Synthase: Evidence for a Dimethylallyl Cation Intermediate in an Aromatic Prenyltransferase Reaction

Dimethylallyltryptophan synthase is an aromatic prenyltransferase that catalyzes an electrophilic aromatic substitution reaction between dimethylallyl diphosphate (DMAPP) and L-tryptophan. The synthase is found in a variety of fungi, where it catalyzes the first committed step in the biosynthesis of the ergot alkaloids. The enzymatic reaction could follow either a dissociative mechanism involving a discrete dimethylallyl cation intermediate or an associative mechanism in which the indole ring directly displaces diphosphate in a single step. In this work, positional isotope exchange (PIX) experiments are presented in support of a dissociative mechanism. When [1-(18)O]-DMAPP is subjected to the synthase reaction and recovered starting material is analyzed, 15% of the (18)O-label is found to have scrambled from a bridging to a nonbridging position on the alpha-phosphorus. Kinetic isotope effect studies show that steps involved in the formation of the arenium ion intermediate are rate-determining, and therefore the scrambling occurs during the lifetime of the dimethylallyl cation/diphosphate ion pair. Similarly, when the unreactive substrate analogue, 6-fluorotryptophan, was employed, complete scrambling of the (18)O-label in DMAPP was observed. To our knowledge, this is the first observation of PIX in any prenyltransferase reaction, and it provides strong evidence supporting the existence of a carbocation intermediate.

Density Functional Study of the In-Line Mechanism of Methanolysis of Cyclic Phosphate and Thiophosphate Esters in Solution: Insight into Thio Effects in RNA Transesterification

Density functional calculations of thio effects on the in-line mechanism of methanolysis of ethylene phosphate (a reverse reaction model for RNA phosphate transesterification) are presented. A total of 12 reaction mechanisms are examined using the B3LYP functional with large basis sets, and the effects of solvation were treated using the PCM, CPCM, and SM5 solvation models. Single thio substitutions at all of the distinct phosphoryl oxygen positions (2', 3', 5', pro-R) and a double thio substitution at the nonbridging (pro-R/pro-S) positions were considered. Profiles for each reaction were calculated in the dianionic and monoanionic/monoprotic states, corresponding to reaction models under alkaline and nonalkaline conditions, respectively. These models provide insight into the mechanisms of RNA transesterification thio effects and serve as a set of high-level quantum data that can be used in the design of new semiempirical quantum models for hybrid quantum mechanical/molecular mechanical simulations and linear-scaling electronic structure calculations.

Examination of P−OR Bridging Bond Orders in Phosphate Monoesters using18O Isotope Shifts in31P NMR

Evidence indicates that phosphate monoesters undergo hydrolysis by a loose transition state with extensive bond fission to the leaving group. It has been proposed that part of the high dependence of the rate on the leaving group pKa (betalg = -1.23) arises from weaker ester bonds in the reactants as the pKa of the leaving group decreases, on the basis of X-ray structures and calculations. In contrast, IR and Raman studies suggest that the leaving group has little effect on the length of the P-OR bridging bond in solution. To gather additional data on this issue, we have used (18)O isotopic shifts in 31P NMR to monitor the bond order of P-O bonds in a range of phosphate esters with different leaving groups. Using this technique, we have been able to evaluate whether significant changes are observed in the P-O bond orders for the bridging and nonbridging positions of methyl, ethyl, phenethyl, propargyl, phenyl, and p-nitrophenyl phosphate using [(16)O(18)O] labeled species in deuterium oxide. The results indicate that the bridging and nonbridging bond orders to phosphorus in phosphate monoesters are not significantly altered by differences in the pKa of the leaving group or by the counterion of the phosphate ester dianion.

Hybrid QM/MM study of thio effects in transphosphorylation reactions: the role of solvation.

Transphosphorylation thio effects in solution are studied using hybrid QM/MM calculations with a d-orbital semiempirical Hamiltonian. Activated dynamics simulations were performed for a 3' ribose-phosphate model in an explicit 20 A sphere of TIP3P water surrounded by a solvent boundary potential, and free energy analysis was performed using the weighted histogram analysis method. Single thio-substitutions at all of the phosphoryl oxygen positions and a double thio-substitution at the nonbridging positions were considered. The reaction free energy profiles are compared with available experimental data, and the role of solvation on the barrier heights and reaction coordinate is discussed. These results provide an important step in the characterization of thio effects in reactions of biological phosphates that may aid in the interpretation of kinetic data and ultimately help to unravel the catalytic mechanisms of ribozymes.

Triple-Quantum Two-Dimensional 27Al Magic Angle Nuclear Magnetic Resonance Study of the Aluminum Incorporation in Calcium Silicate Hydrates

Triple-quantum two-dimensional 27Al magic angle spinning nuclear magnetic resonance (27Al 3Q-MAS NMR) was used to characterize the substitution of Si4+ by Al3+ into the Te−Oc−Te structure of calcium silicate hydrates (C−S−H). This substitution was studied with C−S−H having an Oc/Te ratio of 1 and in equilibrium with Al(OH)3 in aqueous suspensions. In the absence of NaOH, no substitution into the C−S−H structure occurred. Addition of NaOH in the preparation increased the concentration of Al(OH)4- and favored substitution. The deficit of charge resulting from this substitution was compensated by the accommodation of sodium in the interlayer space of the C−S−H. Increasing levels of substituted silicon correspond to higher alkaline and lower calcium contents in the interlayer space. Two substitution sites were distinguished, corresponding to the bridging and nonbridging positions in the chains of tetrahedra. A high Altetra/(Si+Altetra) ratio indicated a redistribution of the aluminum tetrahedral sites to stab...

Structure-Function Relationships of ω-Conotoxin GVIA: SYNTHESIS, STRUCTURE, CALCIUM CHANNEL BINDING, AND FUNCTIONAL ASSAY OF ALANINE-SUBSTITUTED ANALOGUES

The structure-function relationships of the N-type calcium channel blocker, omega-conotoxin GVIA (GVIA), have been elucidated by structural, binding and in vitro and in vivo functional studies of alanine-substituted analogues of the native molecule. Alanine was substituted at all non-bridging positions in the sequence. In most cases the structure of the analogues in aqueous solution was shown to be native-like by 1H NMR spectroscopy. Minor conformational changes observed in some cases were characterized by two-dimensional NMR. Replacement of Lys2 and Tyr13 with Ala caused reductions in potency of more than 2 orders of magnitude in three functional assays (sympathetic nerve stimulation of rat isolated vas deferens, right atrium and mesenteric artery) and a rat brain membrane binding assay. Replacement of several other residues with Ala (particularly Arg17, Tyr22 and Lys24) resulted in significant reductions in potency (<100-fold) in the functional assays, but not the binding assay. The potencies of the analogues were strongly correlated between the different functional assays but not between the functional assays and the binding assay. Thus, the physiologically relevant assays employed in this study have shown that the high affinity of GVIA for the N-type calcium channel is the result of interactions between the channel binding site and the toxin at more sites than the previously identified Lys2 and Tyr13.

The structure of fluorozirconate glasses modified with nitrogen

Abstract Fluorozirconate glasses modified with nitrogen were melted. For basic fluorozirconate glasses of the composition 58 ZrF 4 , 36 BaF 2 , 6 LaF 3 ,10 AlF 3 the aluminium fluoride was replaced by aluminium nitride. The Raman and FT-IR spectra were registered for the studied glasses. Addition of N atoms into the fluorozirconate glass network caused a shift of the bands attributed to vibrations of Zr — F bridging and Zr — F nonbridging groups towards a higher wavenumber. This procedure might cause introduction of the nitrogen atoms into the bridging and nonbridging positions of the fluorine atoms. The nitrogen ions are introduced into the fluorozirconate glasses to improve their mechanical and thermal properties as well as chemical resistance.

The structure of fluorozirconate glasses modified with nitrogen

Fluorozirconate glasses modified with nitrogen were melted. For basic fluorozirconate glasses of the composition 58 ZrF 4, 36 BaF 2, 6 LaF 3,10 AlF 3 the aluminium fluoride was replaced by aluminium nitride. The Raman and FT-IR spectra were registered for the studied glasses. Addition of N atoms into the fluorozirconate glass network caused a shift of the bands attributed to vibrations of Zr — F bridging and Zr — F nonbridging groups towards a higher wavenumber. This procedure might cause introduction of the nitrogen atoms into the bridging and nonbridging positions of the fluorine atoms. The nitrogen ions are introduced into the fluorozirconate glasses to improve their mechanical and thermal properties as well as chemical resistance.

Antisense oligodeoxynucleotides.

Oligodeoxynucleotides have been proposed as both in vitro and in vivo inhibitors of gene expression because of the specificity of Watson-Crick base pair hybridization. Phosphodiester oligodeoxynucleotides (normal DNA) cannot be used as drugs because they are nuclease sensitive. Significant efforts have been made to study phosphorothioate oligodeoxynucleotides, which have a sulfur atom substituted for one of the phosphate oxygen atoms at a nonbridging position. These oligodeoxynucleotides are nuclease resistant, and over the past year they have entered clinical trials. They have also been extensively examined in vitro and have been targeted to the bcr-abl and bcl2 messenger RNAs among others. Methods to maximize the intracellular oligodeoxynucleotide concentration have also been devised. However, significant problems remain, including the significant nonsequence specificity of phosphodiester oligodeoxynucleotides as well as questions of oligodeoxynucleotide uptake into and compartmentalization within cells. An improvement of our understanding of these phenomena is critical to the elaboration of this technology into a clinical therapeutic modality.

Spectroscopic and electrical studies of silver sulfophosphate glasses

New conducting glasses have been synthesized in the xS:(1− x)AgPO 3 system. X-ray diffraction and differential scanning calorimetric studies have confirmed that these glasses can be formed in the range x = 0.0 to 0.35 sulfur content. The glass transition temperature has been found to increase with increase of sulfur content. The dc conductivity, derived from complex impedance measurements, increases with increasing sulfur content and reaches a value of 1.32 × 10 −6 (Ω cm) −1 at room temperature with an activation energy of 0.58 eV in the sample for which x = 0.30. This conductivity is approximately five times higher than that of AgPO 3 glass. Various techniques such as X-ray photoelectron spectroscopy, FT-IR spectroscopy and far-infrared spectroscopy have provided evidence for sulfur participation in the phosphate network by substituting for the oxygen in the non-bridging position. The results demonstrate the formation of [PO 4− x S x ] species and weaker cation-network interactions. The conduction mechanism is discussed is relation to the structure.

3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate

The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated. When [18O]-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi. This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1). No evidence for a covalent enzyme-PEP intermediate could be obtained. No [32P]-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in [18O]-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate. Bromopyruvate inactivated KDO8P synthase in a time dependent process. It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation.

Epimerization in the synthesis of thiophosphoanhydrides

Reaction of ( R p)-[β- 17O]ADPβs or ( S p)-[β- 17O]ADPβs (adenosine 5′,2-thio[2- 17O]diphosphate with R and S configurations, respectively, at P 2) with diphenylchlorophosphate for 15 min in hexamethylphosphoramide followed by aqueous workup produces ( R p)- and ( S p)-P 1-5′-ado-P 3-phenyl-2-thio[2- 17O]triphosphate as an epimeric mixture. Separation of the epimeric coupling products and analysis for bridging and nonbridging 17O reveals that the labeling pattern is similar for products derived from either ( R p)- or ( S p)-[β- 17O]ADPβS, with 17O distributed between bridging and nonbridging positions in both epimers. Similar labeling patterns in the coupling products must have arisen from epimerization at chiral P in ( R p)-[β- 17O]ADPβS and ( S p)-[β- 17O]ADPβS during the initial coupling reaction. Otherwise, one of the coupling products from each stereoisomer would have contained 17O in the P 2-nonbridging position and the other in the P 2-P 3-bridging position. Epimerization at P 2 of [β- 17O]ADPβS could in principle involve either reversible formation of [ 17O]thiometaphosphate or reversible transfer of the [ 17O]thiophosphoryl group to the solvent, hexamethylphosphoramide. Consideration of the rate of epimerization observed in these experiments and the rate of thiometaphosphate formation in solvolysis reactions suggests that epimerization involves solvent participation via the resonance-stabilized zwitterionic species O-[ 17O]thiophosphoryl-hexamethylphosphoramide.

Substrate and inhibitor activities of the screw sense isomers of metal-nucleotide complexes in the formyltetrahydrofolate synthetase reaction.

Phosphorothioate analogues of ATP and isomers of CrATP and CrADP were used to examine the nucleotide stereoselectivity of formyltetrahydrofolate synthetase from procaryotic and eucaryotic sources. Substrate activity of the thio-ATP analogues increased as the site of sulfur substitution was changed from the gamma to the alpha position. Thus, adenine nucleotide analogues substituted with sulfur at an alpha nonbridging position (ATP alpha S isomers) were the most active, and ATP gamma S was inactive. When Mg2+ was used as the divalent cation, both enzymes showed a clear preference (higher V/Km value) for the Sp isomer of ATP beta S although the magnitude of the preference was greater with the bacterial enzyme. With Cd2+ as the divalent cation the Rp isomer was preferred, but the difference was greater with the yeast enzyme. Both (Sp)-MgATP beta S and (Rp)-CdATP beta S have the delta or right-hand screw sense configuration of the metal chelate ring. The reversal of stereoselectivity when the cation was changed indicates that the metal ion is coordinated to the beta-phosphate group. No stereoselectivity was observed when ATP alpha S isomers were used in the presence of Mg2+ or Cd2+, suggesting that the metals are not coordinated to the alpha-phosphate. ATP beta S was also found to be a competitive inhibitor of MgATP and CdATP, and the lowest Ki values were obtained with the lambda screw sense isomers. The screw sense isomers of bidentate CrATP exhibited no detectable substrate activity but were competitive inhibitors of MgATP.(ABSTRACT TRUNCATED AT 250 WORDS)

On the bromine oxidation of hypophosphate: a 31P(18O) positional isotope investigation

Hypophosphate is oxidised quantitatively by bromine in 18O-labelled water to [18O]pyrophosphate; the 18O label is located exclusively in the non-bridging position.

Stereochemical course of DNA hydrolysis by nuclease S1.

Nuclease S1 hydrolyzes the Sp-diastereomer of 5'-O-(2'-deoxyadenosyl)-3'-O-thymidyl phosphorothioate in H2(18)O to [18O]deoxyadenosine 5'-O-phosphorothioate which can be phosphorylated enzymatically to the Sp-diastereomer of [alpha-18O]deoxyadenosine 5'-O-(1-thiotriphosphate). 31P nmr spectroscopy shows the oxygen-18 in this compound to be in a nonbridging position at the alpha-phosphorus, indicating that the hydrolysis reaction catalyzed by nuclease S1 proceeds with inversion of configuration at phosphorus. This result is compatible with a direct nucleophilic attack of H2O at phosphorus without the involvement of a covalent enzyme intermediate.

Template-prime-dependent turnover of (Sp)-dATP alpha S by T4 DNA polymerase. The stereochemistry of the associated 3' goes to 5'-exonuclease.

T4 DNA polymerase converts (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O2]triphosphate) to 2'-deoxyadenosine 5'-O-[18O]-phosphorothioate in the presence of poly(d(A-T).poly(d(A-T)) template-primer. Control experiments involving either omitting the poly(d(A-T)).poly(d(A-T) template-primer or employing the (Rp)-2'-deoxyadenosine 5'-O-(1-thiotriphosphate) diastereomer showed no reaction. It is assumed, therefore, that this conversion as in the P--O case involves incorporation of the thionucleotide into the poly(d(A-T)) followed by hydrolysis resulting from the 3' goes to 5'-exonuclease activity. The 2'-deoxyadenosine 5'-O-[18O] phosphorothioate was converted to (Sp)-2'-deoxyadenosine 5'-O-(1-thio[1-18O]triphosphate), with no change in the configuration at P alpha by using the coupled adenylate kinase-pyruvate kinase enzyme system. A 31P NMR spectrum of the product showed that the 18O was entirely in the nonbridging position, indicating an overall retention in the net turnover process (i.e. incorporation followed by excision). Since the incorporation process involves an inversion of configuration around the phosphorus (Romaniuk, P. J., and Eckstein, F. (1982) J. Biol. Chem. 257, 7684-7688), it must be inferred that the 3' goes to 5'-exonuclease activity of T4 polymerase proceeds with inversion of configuration at the phosphorus atom, most simply via a direct displacement mechanism. This finding represents the first example of phosphodiester hydrolysis catalyzed by an exonuclease that does not involve a covalent phosphoryl-enzyme intermediate (Knowles, J. R. (1980) Annu. Rev. Biochem. 49, 877-919).