1H NMR titrations of hydroxy protons in aqueous solution as a method of investigation of intramolecular hydrogen-bonding in phosphorylated compounds: examples of myo-inositol 2-phosphate and myo-inositol 1,2,6-tris(phosphates)

1H NMR hydroxy proton titration experiments for myo-inositol 2-phosphate and myo-inositol 1,2,6-tris(phosphates) in aqueous solution are presented to demonstrate that by following OH signals versus pH, evidence can be brought for HB interaction between the hydroxyl and phosphate groups. The chemical shifts of the OH protons vicinal to phosphate groups appear deshielded by ca. 2.5 ppm with regard to those two centers removed from the phosphates. Remarkably, the deshielded protons are only present when their neighboring phosphate groups are fully deprotonated but persist until high pHs. From these results, C-OH...2-O3P-O type I, C-HO...-HO3P-O type II and C-OH...-HO3P-O type III hydrogen bonds are evidenced and discussed.     

CHARMM force field parameters for simulation of reactive intermediates in native and thio-substituted ribozymes

Force field parameters specifically optimized for residues important in the study of RNA catalysis are derived from density-functional calculations, in a fashion consistent with the CHARMM27 all-atom empirical force field. Parameters are presented for residues that model reactive RNA intermediates and transition state analogs, thio-substituted phosphates and phosphoranes, and bound Mg(2+) and di-metal bridge complexes. Target data was generated via density-functional calculations at the B3LYP/6-311++G(3df,2p)// B3LYP/6-31++G(d,p) level. Partial atomic charges were initially derived from CHelpG electrostatic potential fitting and subsequently adjusted to be consistent with the CHARMM27 charges. Lennard-Jones parameters were determined to reproduce interaction energies with water molecules. Bond, angle, and torsion parameters were derived from the density-functional calculations and renormalized to maintain compatibility with the existing CHARMM27 parameters for standard residues. The extension of the CHARMM27 force field parameters for the nonstandard biological residues presented here will have considerable use in simulations of ribozymes, including the study of freeze-trapped catalytic intermediates, metal ion binding and occupation, and thio effects.     

ZrIV-tetraphenylporphyrinates as nuclease mimics: structural, kinetic and mechanistic studies on phosphate diester transesterification

The Zr(IV)-tetraphenylpor-phyrinates Zr(TPP)(X,X'), (X,X' = -OAc, -OMe, Cl ) 4-6, 8 were prepared and their complexing properties as well as catalytic properties towards solvolysis of the phosphate diesters hpp (2), dmp (3) and pmp (16) characterised. The diesters 2 and 16, representing model phosphates for RNA and DNA, were substrates for the catalyst Zr(TPP)Cl2 (4), and rate accelerations over background by 6-9 orders of magnitude were measured. These accelerations are comparable to those of dinuclear transition metal catalysts and lanthanide ions. Catalytic turnover was observed. Kinetic studies revealed that the catalytically active species of 4 in the solvolysis of 2 and 16 in methanol-containing solvents are dinuclear complexes containing either one or two phosphate esters depending upon the phosphate concentration. Besides the usual solvolysis pathway of the RNA model hpp (2), which proceeds via the cyclophosphate 20, a second, unusual pathway via direct substitution of the hydroxypropyl substituent was found. X-ray analysis of the Zr(TPP)(dmp) complex 19 revealed a dinuclear structure with two bridging dmp ligands and one monomethyl phosphate unit. In 19 one of the two dmp residues occurs in a very unusual high energy ac,ap conformation. Based on this structure and on the kinetic data, mechanistic models for the two solvolysis reaction pathways were developed. From an extensive CSD search on phosphodiester structures no correlation between P-O ester bond lengths and diester conformations could be found. However, P-O ester bonds decrease in length with increasing formal charge of the complexing metal ions. This underlines the higher importance of electrostatic activation relative to stereoelectronic effects in phosphodiester hydrolysis.     

Smooth solvation method for d-orbital semiempirical calculations of biological reactions. 1. Implementation

The present paper describes the extension of a recently developed smooth conductor-like screening model for solvation to a d-orbital semiempirical framework (MNDO/d-SCOSMO) with analytic gradients that can be used for geometry optimizations, transition state searches, and molecular dynamics simulations. The methodology is tested on the potential energy surfaces for separating ions and the dissociative phosphoryl transfer mechanism of methyl phosphate. The convergence behavior of the smooth COSMO method with respect to discretization level is examined and the numerical stability of the energy and gradient are compared to that from conventional COSMO calculations. The present method is further tested in applications to energy minimum and transition state geometry optimizations of neutral and charged metaphosphates, phosphates, and phosphoranes that are models for stationary points in transphosphorylation reaction pathways of enzymes and ribozymes. The results indicate that the smooth COSMO method greatly enhances the stability of quantum mechanical geometry optimization and transition state search calculations that would routinely fail with conventional solvation methods. The present MNDO/d-SCOSMO method has considerable computational advantages over hybrid quantum mechanical/molecular mechanical methods with explicit solvation, and represents a potentially useful tool in the arsenal of multi-scale quantum models used to study biochemical reactions.     

Chemistry of oxaphosphoranes (review)

The research of recent years on the synthesis of new types of phosphoranes with P-O bonds (oxaphosphoranes) is examined. Particular attention is directed to the problems of the thermodynamic stabilities of oxaphosphoranes and the homolytic reactions of hydrooxaphosphoranes.Translated from Khimiya Geterotsiklicheskikh Soedinenii, No. 9, pp. 1155–1171, September, 1989.     

Benchmark calculations of proton affinities and gas-phase basicities of molecules important in the study of biological phosphoryl transfer

Benchmark calculations of proton affinities and gas-phase basicities of molecules most relevant to biological phosphoryl transfer reactions are presented and compared with available experimental results. The accuracy of proton affinity and gas-phase basicity results obtained from several multi-level model chemistries (CBS-QB3, G3B3, and G3MP2B3) and density-functional quantum models (PBE0, B1B95, and B3LYP) are assessed and compared. From these data, a set of empirical bond enthalpy, entropy, and free energy corrections are introduced that considerably improve the accuracy and predictive capability of the methods. These corrections are applied to the prediction of proton affinity and gas-phase basicity values of important biological phosphates and phosphoranes for which experimental data does not currently exist. Comparison is made with results from semiempirical quantum models that are commonly employed in hybrid quantum mechanical/molecular mechanical simulations. Data suggest that the design of improved semiempirical quantum models with increased accuracy for relative proton affinity values is necessary to obtain quantitative accuracy for phosphoryl transfer reactions in solution, enzymes, and ribozymes.     

1H NMR spectra,dipole moments,and conformations of 3-oxo-6,7-benzo-1,5,3-dioxaphosphepines

¹H NMR spectroscopy, measurements of dipole moments, and the Kerr effect have been used to study the conformational structure in solutions of 3-X-3-oxo-6,7-benzo-1,5,3-dioxaphosphepines (X=Me, Et, Ph, Cl, NEt₂, OEt, OPh). On the basis of x-ray diffraction data, by means of the Dillen-Geise method, possible conformers of the seven-membered ring have been described quantitatively: chair, twist, and twist-boat. It has been shown that the compounds are characterized by a three-component equilibrium of chair and flexible forms. The relative populations of the conformers depend on the nature of the substituent X. The relationships in internal rotation around the P-Ph and P-O(R) bonds are discussed.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 7, pp. 1517–1523, July, 1991.     

Order-disorder in the super-sodalite Zn3Al6(PO4)12, 4tren, 17H2O (MIL-74): a combined XRD-NMR assessment

A new mixed zinc-aluminum phosphate Zn(3)Al(6)(PO(4))(12), 4tren, 17H(2)O (MIL-74) has been hydrothermally synthesized with the tris(2-aminoethyl)amine (tren) as a structure-directing agent (453 K, 36 h, autogenous pressure). The solid was characterized by a nonclassical method combining single-crystal X-ray diffraction and several solid-state NMR experiments, RFDR, C7 double quantum ((31)P), and 3QMAS ((27)Al). Its crystal structure is cubic, a = 16.7942(1) A, but the choice of the space group does not follow usual routes of structure determination, due to some "disorder" between Zn and Al. It can be assigned as well to I-43m or to P-43n. The open-framework is built up from an enneameric unit (T = Zn, Al) containing five TO(4) and four PO(4) tetrahedra (one of the P-O bonds is terminal). A central TO(4) tetrahedral unit shares all of the corners with four phosphates groups. Two phosphate groups are connected to two other peripheral TO(4) units. It results in the formation of a "pseudo" planar building block T(5)P(4) consisting of four square 4-rings. The connection of the T(5)P(4) units generates a three-dimensional framework, which defines a super-sodalite topology. The resulting cavities (diameter of 10 A) are bound by 12-ring windows in which are located the tren species in interaction with the phosphate groups (mainly terminal P-O bonds) through hydrogen bonds. A cluster of 17 water molecules occupies the center of the super-sodalite cage. The cationic (Zn, Al) occupancy is discussed for this specific topology.     

Transition state differences in hydrolysis reactions of alkyl versus aryl phosphate monoester monoanions

Although aryl phosphates have been the subject of numerous experimental studies, far less data bearing on the mechanism and transition states for alkyl phosphate reactions have been presented. Except for esters with very good leaving groups such as 2,4-dinitrophenol, the monoanion of phosphate esters is more reactive than the dianion. Several mechanisms have been proposed for the hydrolysis of the monoanion species. (18)O kinetic isotope effects in the nonbridging oxygen atoms and in the P-O(R) ester bond, and solvent deuterium isotope effects, have been measured for the hydrolysis of m-nitrobenzyl phosphate. The results rule out a proposed mechanism in which the phosphoryl group deprotonates water and then undergoes attack by hydroxide. The results are most consistent with a preequilibrium proton transfer from the phosphoryl group to the ester oxygen atom, followed by rate-limiting P-O bond fission, as originally proposed by Kirby and co-workers in 1967. The transition state for m-nitrobenzyl phosphate (leaving group pK(a) 14.9) exhibits much less P-O bond fission than the reaction of the more labile p-nitrophenyl phosphate (leaving group pK(a) = 7.14). This seemingly anti-Hammond behavior results from weakening of the P-O(R) ester bond resulting from protonation, an effect which calculations have shown is much more pronounced for aryl phosphates than for alkyl ones.     

Negative hyperconjugation in phosphorus stabilized carbanions

The electronic Fukui function is used to give qualitative electronic proof on the existence of back-bonding from the carbon lone pair toward the sigma* P-Y and P-O orbitals in phosphorus stabilized carbanions. NBO analyses are used to investigate the energetic, electronic, and structural impacts of this negative hyperconjugation interaction. The observed energetic stabilization can indeed be attributed to the electronic delocalization of the lone pair toward the antibonding orbitals. This delocalization is furthermore responsible for the shorter P-C bonds, longer P-Y (P-O) bonds, and wider Y-P-Y angles observed for the anionic compounds compared to their neutral counterparts. From the electronic NBO analysis it becomes clear that phosphorus containing functional groups are best described as sigma donor/pi acceptors.     

Enantioselective catalysis using phosphorus-donor ligands containing two or three P-N or P-O bonds

This review describes some recent developments in the area of asymmetric catalysis using organometallic complexes of ligands which contain two or three P-O or P-N bonds. This category of ligands has, until the last five years or so, been underrepresented in asymmetric catalysis, particularly in contrast to phosphine ligands. Recent breakthroughs in methodology for the synthesis and manipulation of such materials have resulted in the delivery of ligands which demonstrate remarkably high enantioinduction properties in a series of asymmetric transformations.     

Unifying the Current Data on the Mechanism of Cleavage–Transesterification of RNA

The various mechanisms by which RNA is hydrolyzed are currently under intense investigation. The first step in the hydrolysis pathway is a cleavage–transesterification in which a 2′-OH group attacks a 3′,5′-phosphodiester linkage with departure of the 5′ group. The second step involves the opening of a 2′,3′-cyclic phosphodiester. Complications in these steps arise from multiple possible pathways involving specific acid and base as well as general acid and base catalysis. In addition, controversy exists concerning the protonation states of the phosphodiesters and any intermediate phosphoranes under various experimental conditions. A summary of mechanistic studies involving general and specific acid/base catalysis of RNA hydrolysis and the hydrolysis of RNA analogs is presented herein, along with the interpretations given by the original authors. Included are theoretical calculations, kinetic studies, pK_a determinations, isotope effects, Hammett and Brønsted correlations, and model studies. Recent analyses of the mechanism of RNase A are also briefly reviewed. Two limiting mechanisms for the cleavage–transesterification step that unify the data in the literature and differ only in the role of the phosphorane and its protonation state are given at the outset. An analysis of the literature studies supporting these mechanisms is then provided.     

Synthesis, structure and isomerization of arylphosphoranes with anti-apicophilic bonding modes using a novel bidentate ligand with two C(2)F(5) groups

A series of anti-apicophilic pentacoordinate phosphoranes (with one chelating substituent in an O-equatorial, C-apical bonding mode at pentacoordinated phosphorus atom) bearing a para-substituted aryl group (-C(6)H(4)(p-X); X = H, CF(3), F, OMe) or a mesityl (2,4,6-trimethylphenyl) group were isolated using a novel bulky bidentate ligand with two C(2)F(5) groups. These phosphoranes were stable to isomerization at room temperature, and quantitatively converted into the corresponding more stable isomers (O-apical) at elevated temperatures in solution. On the basis of a kinetic study, the free energy of activation (DeltaG(double dagger)) of the stereomutation of the O-equatorial mesitylphosphorane to its O-apical isomer was higher than that of the CF(3) derivative by 2.6 kcal mol(-1), giving rise to a further example of the steric effect of the C(2)F(5) group to freeze the isomerization of the pentacoordinate phosphorus compounds. Kinetic measurements of the isomerization of the O-equatorial ortho-unsubstituted derivatives (-C(6)H(4)(p-X)) to the corresponding O-apical isomers suggested that the O-equatorial isomers were stabilized by the pi --> sigma*(P-O) interaction in the ground state.     

The colorimetric determination of orthophosphate in the presence of condensed phosphates

The linear condensed phosphates inhibit the development of color due to orthophosphatc by the method of LOWRY AND LOPEZ. The higher members of the scries (the polymetaphosphates) have more effect than the lower members (pyrophosphate, tripoly-and tetrapolyphosphates). The two cyclic metaphosphates, trimeta- and tetrametaphosphate, do not inhibit. The inhibition by polymetaphosphate is partly overcome by storing the developed color for about 90 min. Low concentrations of copper and nickel and higher concentrations of iron(III) remove either partially or completely the inhibition due to polymetaphosphates. None of the metaphosphates interferes in the estimation of orthophosphate by the isobutanol extraction method of BERENBLUM AND CHAIN     

Hybrid QM/MM study of thio effects in transphosphorylation reactions

Results of a series of hybrid quantum mechanical/molecular mechanical (QM/MM) activated dynamics simulations of thio effects in the transphosphorylation (methanolysis) of a 2'-ribose, 5'-methyl phosphate-diester under basic conditions are presented. Single and double substitutions in the nonbridging oxygen positions exhibit thio effects in accord with experimental data and show the existence of a stable intermediate. Thio substitution at the 2' and 5' positions resulted in reactions having a single transition state with increased and decreased free energy barriers, respectively, relative to the unsubstituted reaction. In all of the reactions except for the 5' substitution, the rate-limiting step corresponds to exocyclic cleavage. In the 5' substitution reaction, the rate-limiting step corresponds to endocyclic cleavage and shows a considerable reverse thio effect, in accord with experimental observations of phosphates with enhanced leaving groups. Thio substitution at the 3' position results in a mild reverse thio effect that arises from electronic stabilization of the dianionic transition state. The results presented here provide an important step toward the development and application of new hybrid QM/MM methods that, combined with experiment, may provide a detailed picture of the molecular mechanisms of RNA catalysis.     

P-O donor action from carboxylate anions with phosphorus in the presence of hydrogen bonding. A model for phosphoryl-transfer enzymes

A series of phosphorus compounds (1-3) containing anionic carboxylate groups were synthesized by treatment of the respective neutral precursor acid forms B-D with amines, which also served to introduce hydrogen-bonding interactions. The compounds, subjected to X-ray structure analysis, resulted in hexacoordinated anionic phosphoranates 1A and 1B, a pseudo-trigonal-bipyramidal anionic phosphine (2), and a trigonal-bipyramidal anionic phosphine oxide (3). The structures revealed that P-O donor coordination was present in all members of the anionic series 1-3 and resulted in stronger interactions than existed in the precursor neutral acid forms B-D as measured by the presence of shorter P-O distances. Evaluation of the energies of the donor interactions relative to the energies of the hydrogen bonds that were present showed that the donor energies now exceeded the hydrogen bond strengths. (31)P chemical shifts indicated that the basic coordination geometries were retained in solution. Both 1A and 1B are chiral and exist as racemates. The results suggest that mechanisms of phosphoryl-transfer enzymes should benefit by taking into account donor interactions at phosphorus by residues at active sites in addition to the inclusion of hydrogen bonding. Reference is made to specific phosphoryl-transfer enzymes.     

Ab initio studies of conformational properties of dimethyl diphosphate dianion and its complex with magnesium

The conformational properties of the diphosphate linkage have been studied with ab initio methods using the dimethyl diphosphate dianion (1) and magnesium dimethyl diphosphate (2) as models. The ab initio energy and geometry of the conformers around the P-O bonds have been determined at the self-consistent-field (SCF) using the 6-31G* and the tzp basis sets; whereas, the 6-31G* basis set alone has been used for 2. In addition, the adiabatic connection method (ACM) of density functional theory (DFT) using the dzvp basis set has been employed for 1. The optimization of all possible staggered conformers assumed for the four P-O bonds, led to nine minima for 1. In agreement with the general anomeric effect, the sc conformation about the P-O bonds is clearly preferred over the ap one. Vibrational frequencies were calculated at the SCF level using the 6-31G* basis set and used to evaluate zero-point energies, thermal energies, and entropies for all minima of 1. The effect of zero-point energies and thermal energies is quite small. However, the effect of entropies, mainly resulting from a multiplicity contribution, changes the stability of the conformers. For each minimum of 1, up to six different arrangements of the Mg²⁺ were used to determine minima of 2. This procedure led to 21 distinct minima. The presence of the magnesium counter-ion appeared to completely change the structure and relative energy of the conformers. The preferred structures of the complex exhibit the (sc, ap) orientation around the two central P–O bonds and an arrangement in which the magnesium cation is coordinated by three phosphoryl oxygen atoms. The results of this work clearly demonstrate that interactions with the metal counter-ion can induce conformational changes in the overall 3D-shape adopted by molecules containing diphosphate linkages. The PM3 and MNDO quantum semi-empirical methods and molecular mechanics methods using the CVFF force field were tested and large differences in the minimum structures, as well as in the conformational energies between these and ab initio methods, are discussed.     

Vibrational spectra of four alkaline earth cyclo-hexaphosphates

FT-IR and FT-Raman spectra of four alkaline earth (Mg, Ca, Sr and Ba) cyclo-hexaphosphates have been recorded and analysed. FT-Raman spectra of the deuterated analogues of these compounds are used to clear the ambiguity regarding the OH vibrations in the low frequency region. The spectra reveal that the P6O18(6) anion ring in all compounds have independent PO4 tetrahedra present in it. The P-O(P)and P-O(M')(M'-Mg, Ca, Sr and Ba) bonds become stronger as the cation size decreases. The P6O18(6-) anion ring is distorted in the Mg and Ca compounds. In the Sr and Ba compounds, free and hydrogen bonded water molecules of varying strength are present, whereas in Mg and Ca compounds no free water molecules exist. The POP bridge angle of the Mg, Ca and Sr compounds are estimated using the correlations between the POP bridge stretching frequencies and the bridge angle value.     

Synthesis and characterization of linear, hyperbranched, and dendrimer-like polymers constituted of the same repeating unit

The synthesis of a linear polymer that includes both P=N and P=S double bonds, and P-O and P-C single bonds is reported by using two different paths that involve deprotection reactions and the Staudinger reaction. The preparation of hyperbranched polymers made up of OC6H4P(Ph)2=N-P=S repeating units is also described. Five generations of dendrimers originating from the same building blocks were prepared. The characterisation of all these phosphorus-based macromolecular architectures (solution behaviour, size exclusion chromatography, intrinsic viscosity, thermal behaviour) revealed marked differences in their respective behaviour.