R. Stan Brown

Mechanistic studies of La3+- and Zn2+-catalyzed methanolysis of aryl phosphate and phosphorothioate triesters. Development of artificial phosphotriesterase systems

The methanolyses of a series of O,O-diethyl O-aryl phosphates (2,5) and O,O-diethyl S-aryl phosphorothioates (6) promoted by methoxide and two metal ion systems, (La3+)2(-OCH3)2 and 4:Zn2+:-OCH3 (4 = 1,5,9-triazacyclododecane) has been studied in methanol at 25 degrees C. Brønsted plots of the logk2 values vs. pKa for the phenol leaving groups give beta(lg) values of -0.70, -1.43 and -1.12 for the methanolysis of the phosphates and -0.63, -0.87 and -0.74 for the methanolysis of the phosphorothioates promoted by the methoxide, La3+ and Zn2+ systems respectively. The kinetic data for the metal-catalyzed reactions are analyzed in terms of a common mechanism where there is extensive cleavage of the P-XAr bond in the rate-limiting transition state. The relevance of these findings to the mechanism of action of the phosphotriesterase enzyme is discussed.

Metal-catalyzed alcoholysis reactions of carboxylate and organophosphorus esters

Publisher Summary This chapter elaborates the metal ion-catalyzed hydrolytic reactions of esters, amides, and organophosphorus esters. Apart from being fundamentally interesting and industrially important, phosphoryl and acyl transfer reactions are the key biological processes. Virtually, all the transition metal and lanthanide ions that have been investigated have some catalytic activity in promoting the transesterification of carboxylate esters and neutral phosphate esters. The early studies of metal-catalyzed acyl transfer reactions were predicated on the idea that a reduced polarity/dielectric constant medium would allow one to better realize the catalytic potential of the metal ion by reducing the tightness of the solvation shell around the metal ion and its constituents, as well as allow a stronger interaction energy with substrate. Despite a great deal of effort that has led to an increased understanding of how enzymatic catalysis might occur, it is generally held that none of the several models so far described approaches the enormous catalytic efficiency of natural enzymes. The ideas being discussed in this chapter will spur further work using the multiple effects of structure and medium to bring us closer to understanding the ways, in which nature performs such transformations.

Investigation of the effect of oxy bridging groups in dinuclear Zn(II) complexes that catalyze the cleavage of a simple phosphate diester RNA analogue.

Two sets of dinuclear Zn(II) complexes were prepared to determine the effect of the presence of oxyanionic bridging groups between the metal centers on the catalytic activity toward the methanolysis of the RNA analogue 2-hydroxypropyl-4-nitrophenyl phosphate (HPNPP, 2). The Zn(II)2 complexes of bis(di-(2-pyridylmethyl)amino)-m-xylene (6) and 2,6-bis(di-(2-pyridylmethyl)amino)-4-methylphenol (7) were compared to assess the effect of a bridging phenoxide ligand, while the Zn(II)2 complex of 1,3-bis-N1-(1,5,9-triazacyclododecyl)-propan-2-ol (8) was prepared to determine the effect of the 2-propoxy group compared to the previously studied complex of 1,3-bis-N1-(1,5,9-triazacyclododecyl)-propane (4). Detailed kinetic studies of the cleavage of 2 including k(obs) vs [catalyst] plots and (s)(s)pH-rate profiles were performed for each system along with potentiometric titration experiments to determine the acid dissociation constants for the catalytically relevant groups. The results show that inclusion of the phenoxy bridging group in 7:Zn(II)2 reduces the second-order catalytic rate constant (k2(cat)) for cleavage of 2 by a factor of 160 relative to that of 6:Zn(II)2, while the incorporation of a propoxy group in 8:Zn(II)2 reduces its efficacy by 3.7 x 10(4) times relative to 4:Zn(II)2. Energetics calculations reveal that 6:Zn(II)2 offers a 3.7 kcal/mol greater stabilization of the reaction transition state for the cleavage of 2 than does 7:Zn(II)2 and that 4:Zn(II)2 affords 6.5 kcal/mol greater transition state stabilization than does 8:Zn(II)2. The analyses show that the reduction in the transition state stabilization experienced with the complexes having permanently bridging oxyanion groups stems almost entirely from a weaker binding of the phosphate and catalyst, and a reduced catalytic rate constant. These results indicate that the presence of a bridging oxyanion ligand between the metal centers, a common structural element required for the successful formation of many small molecule dinuclear catalysts that show cooperative activity in water, significantly impairs the catalytic efficiency for cleavage of 2.

Cu(II)-Mediated decomposition of phosphorothionate PS pesticides. Billion-fold acceleration of the methanolysis of fenitrothion promoted by a simple Cu(II)–ligand system

The kinetics of methanolysis of the title compound (3) were studied in the presence of Cu(2+), introduced as Cu(OTf), in the presence of 0.5-1.0 eq. of methoxide and in the presence of 1.0 eq. of a ligand such as bipyridyl (5), phenanthroline (6) or 1,5,9-triazacyclododecane (4). In all cases the active species involve Cu(2+)((-)OCH(3)). In the case of added strong-binding ligands 5 or 6, a plot of the observed rate constant for methanolysis of 3 vs. [Cu(2+)](total) gives a curved line modelled by a process having a [Cu(2+)](1/2) dependence consistent with an active monomeric species in equilibrium with an inactive dimer i.e.[LCu(2+)((-)OCH(3))](2) <==> 2LCu(2+)((-)OCH(3)). In the case of the added strong binding ligand 4, the plot of the observed rate constant for methanolysis of 3 vs.[Cu(2+)](total) gives a straight line consistent with the catalytically active species being Cu(2+)(OCH(3)) which shows no propensity to form inactive dimers. Turnover experiments where the [3] > [Cu(2+)](total) indicate that the systems are truly catalytic. In the optimum case a catalytic system comprising 1 mM of the complex 4Cu(2+)((-)OCH(3)) catalyzes the methanolysis of 3 with a t(1/2) of approximately 58 s accounting for a 1.7 x 10(9)-fold acceleration relative to the background reaction at near neutral (s)(s)pH (8.75).

Biomimetic Cleavage of RNA Models Promoted by a Dinuclear Zn(II) Complex in Ethanol. Greater than 30 kcal/mol Stabilization of the Transition State for Cleavage of a Phosphate Diester

The cleavage of a series of seven substituted aryl 2-hydroxypropyl phosphates (1a-g) promoted by a dinuclear Zn(II) complex (3:Zn(II)2:(-OCH2CH3)) was investigated in ethanol at pH 9.0 +/- 0.2 and 25 degrees C. The kinetics for appearance of the product phenols follow very strong saturation behavior for all substrates where the dissociation constant of the bound complex has an upper limit of Km = 3 x 10(-7) M and the k(cat)(max corr.) values (corrected for triflate inhibition) range from 168 to 3 s(-1). A partial s(s)pH/log k(cat)(max corr). profile for the 3:Zn(II)2:(-OCH2CH3)-catalyzed reaction of le (3-methoxyphenyl 2-hydroxypropyl phosphate) is bell-shaped, plateauing from 7.9-10, and is fit to a two kinetically important ionizations having s(s)pKa values of 7.22 and 10.9. The Brønsted plot of log (k(cat)(max corr.)) vs. the s(s)pKa values for the phenols shows a break at about 14.3 with two beta(lg), values of -1.12 and 0.0. This is analyzed in terms of a change in rate limiting step from cleavage of the phosphate to a conformational change where the binding of the phosphate changes from one P-O- ----Zn(II) interaction to a Zn(II)----O-P-O---Zn(II) double activation. An energetics calculation comparing the ethoxide promoted cleavage of 1a-g with the 3:Zn(ll)2:(-OEt) promoted reaction indicates that the complex, 3:Zn(II)2, stabilizes the ethoxide plus substrate transition state for the cleavage of 1a-g by between 33 and 36 kcal/mol. The origins of the large stabilization are discussed in terms of the effect of the medium on the various rate and equilibrium constants involved.

Cleavage of an RNA model catalyzed by dinuclear Zn(II) complexes containing rate-accelerating pendants. Comparison of the catalytic benefits of H-bonding and hydrophobic substituents.

The transesterification of a simple RNA model, 2-hydroxypropyl p-nitrophenyl phosphate (2, HpNPP) promoted by seven dinuclear Zn(II) catalysts (3,4,5,6,7,8,9:Zn(II)2:(-OCH3)) based on the bis[bis(2-substituted-pyridinyl-6-methyl)]amine ligand system was investigated in methanol under sspH-controlled conditions at 25.0 ± 0.1 °C. The two metal complexing ligands were joined together via the amino N connected to a m-xylyl linker (3, 4, 5, 6, 7) where the 2-pyridinyl substituent = H, CH3, (CH)4, NH2, and NH(C═O)CH3, respectively, and a propyl linker (8, 9) where the ring substituent = H and CH3. All of the dinuclear complexes except 8:Zn(II)2 exhibit saturation kinetics for the kobs versus [catalyst] plots from which one can determine catalyst:substrate binding constants (KM), the catalytic rate constants for their decomposition (kcat), and the second order catalytic rate constants (k2cat = kcat/KM). In the case of 8:Zn(II)2, the plots of kobs versus [catalyst] as a function of sspH are linear, and the catalytic rate constants (k2cat) are defined as the gradients of the plots. Analysis of all of the data at the sspH optimum for each reaction indicates that the presence of the amino and acetamido H-bonding groups and the CH3 group provides similar increases of the kcat terms of 25−50 times that exhibited by the parent complex 3:Zn(II)2. However, in terms of substrate catalyst binding (KM), there is no clear trend that H-bonding groups or the CH3 group provides stronger binding than the parent complex. In terms of the overall second order catalytic rate constant, the CH3, amino, and NH(C═O)CH3 groups provide 20, 10, and 68 times the k2cat observed for the parent complex. In the case of 9:Zn(II)2, the presence of the methyl groups provides a 1000-fold increase in activity (judged by k2cat) over the parent complex 8:Zn(II)2. The results are interpreted to indicate that H-bonding effects may be important for catalysis and less so for substrate binding, but the steric effect and impact on the local polarity provided by a methyl substituent is just as effective and in fact may form part of the acceleratory effect attributed to H-bonding in related systems.

Leaving Group Assistance in the La3+-Catalyzed Cleavage of Dimethyl (o-Methoxycarbonyl)aryl Phosphate Triesters in Methanol

The catalytic methanolysis of a series of dimethyl aryl phosphate triesters where the aryl groups contain an o-methoxycarbonyl (o-CO2Me) substituent (4a-i) was studied at 25 degrees C in methanol containing La3+ at various concentrations and (s)(s)pH. Determination of the second-order rate constant for La3+(2)-catalyzed cleavage of substrate 4a (dimethyl (o-methoxycarbonyl)phenyl phosphate) as a function of (s)(s)pH was assessed in terms of a speciation diagram that showed that the process was catalyzed by La3+(2)(-OCH3)x dimers, where x = 1-5, that exhibit only a 5-fold difference in activity between all the species. The second-order catalytic rate constants (k2(La)) for the catalyzed methanolysis of 4a-i at (s)(s)pH 8.7 fit a Brønsted relationship of log k2(La) = (-0.82 +/- 0.11)(s)(s)pKa(lg) + (11.61 +/- 1.48), where the gradient is shallower than that determined for a series of dimethyl aryl phosphates that do not contain the o-CO2Me substituent, log k2(La) = (-1.25 +/- 0.06)(s)(s)pKa(lg) + (16.23 +/- 0.75). Two main observations are that (1) the o-CO2Me group preferentially accelerates the cleavage of the phosphate triesters with poor leaving groups relative to those with good leaving groups and (2) it provides an increase in cleavage rate relative to those of comparable substrates that do not have that functional group, e.g., k2(La)(dimethyl o-(methoxycarbonyl)phenyl phosphate)/k2(La)(dimethyl phenyl phosphate) = 60. Activation parameters for the La3+(2)-catalyzed methanolysis of 4a and dimethyl 4-nitrophenyl phosphate show respective DeltaH(double dagger) (DeltaS(double dagger)) values of 3.3 kcal/mol (-47 cal/mol x K) and 0.7 kcal/mol (-46.5 cal/mol x K). The data are analyzed in terms of a concerted reaction where the catalytic complex (La3+(2)(-OCH3)(x-1)) binds to the three components of a rather loose transition state composed of a nucleophile CH3O-, a nucleofuge -OAr, and a central (RO)2P(2+)-O(-) in a way that provides leaving group assistance to the departing aryloxy group.

Comparison of Cu(II)-Promoted Leaving Group Stabilization of the Cleavage of a Homologous Set of Phosphate Mono-, Di-, and Triesters in Water, Methanol, and Ethanol

The cleavage of a set of phosphate mono-, di-, and triesters having a Cu(II)-complexed 2-phenanthrolyl group at the ortho-position of a departing phenoxide was studied in water and ethanol. Experimentally observed pH/rate profiles, solvent deuterium kinetic isotope effects, and activation parameters are compared with those obtained in methanol. The pH/rate profile in each solvent exhibits an extended plateau due to solvent attack on forms designated as [Cu(II):1b/c](0) for the monoester, [Cu(II):2b](+), for the diester, and [Cu(II):3a](2+) for the triester. The solvent dkie values (k(H)/k(D)) for the three complexes are 0.91, 0.95, and 0.83 for decomposition of [Cu(II):1b/c](0) in water (W), methanol (M), and ethanol (E), 1.22, 1.09, and 1.29 for [Cu(II):2b](+) in W, M, and E, and 1.94, 2.2, and 1.96 for [Cu(II):3a](2+) in W, M, and E. Near unit, or slightly inverse values for the monoester are taken as evidence for little involvement of solvent in a highly dissociative TS for P-OAr cleavage, with slightly higher solvent dkie values for the diester signifying the onset of some solvent participation in assisting the nucleophilic displacement. The larger primary dkie for the triester gives evidence for a solvent-assisted delivery of ROH in the cleavage through a more associative mechanism. Activation parameters for each substrate in the solvents are compared, indicating that the transition from methanol to ethanol for each substrate involves a near cancellation of the ΔΔH(‡) and -TΔΔS(‡) values (25 °C) so that the respective rates in both solvents are very similar. The transition from alcohol to water produces variable effects, with ΔΔH(‡) and -TΔΔS(‡) values canceling for cleavage of the triester and being additive for the mono and diester, explaining their 100-500 rate reduction in passing from methanol to water. The rate enhancing effects of the Cu(II)-promoted leaving group assistance in all three solvents are substantial and estimated at 10(12)-10(15) for the monoester, 10(12)-10(14) for the diester, and 10(5) for the triester relative to their background reactions.

A Study of the Kinetics of La3+-Promoted Methanolysis of S-Aryl Methylphosphonothioates: Possible Methodology for Decontamination of EA 2192, the Toxic Byproduct of VX Hydrolysis

The kinetics of the La3+-catalyzed methanolysis of a series of S-aryl methylphosphonothioates (4a-e, phenyl substituents = 3,5-dichloro, 4-chloro, 4-fluoro, 4-H, 4-methoxy) were studied at 25 °C with s(s)pH control. The reaction involves saturation binding of the anionic substrates to dimeric La3+/methoxide catalysts formulated as La2(3+)(-OCH3)x, where x = 2-5 depending on the solution s(s)pH. Cleavage of the La3+-bound methylphosphonothioates is fast, ranging from 5 × 10(-3) s(-1) to 5.5 × 10-(5) s(-1) for substrates 4a-e at a s(s)pH of 8.4 and 1.6 × 10(-1) s(-1) to 4 × 10(-3) s(-1) at a s(s)pH of 11.7. The rate accelerations for the methanolysis of substrates 4a-e, relative to their background methoxide-promoted reactions, average 7 × 10(10) and 1.5 × 10(9), respectively, at s(s)pHs of 8.4 and 11.7. The catalytic system is predicted to cleave EA 2192 (S-2(N,N-di-iso-propylaminoethyl)methylphosphonothioate), a toxic byproduct of the hydrolysis of VX, with a t1/2 between 4 and 8 min at a s(s)pH of 8.4, and 27 min at a s(s)pH of 11.7.

Mechanistic studies of La3+ and Zn2+-catalyzed methanolysis of O-ethyl O-aryl methylphosphonate esters. An effective solvolytic method for the catalytic destruction of phosphonate CW simulants

The kinetics of methanolysis of six O-ethyl O-aryl methylphosphonates (6a-f) promoted by methoxide, La3+ and 1,5,9-triazacyclododecane complex of Zn2+(-OCH3) (5:Zn2+(-OCH3)) were studied as simulants for chemical warfare (CW) agents, and analyzed through the use of Brønsted plots. The beta(lg) values are, respectively, -0.76, -1.26 and -1.06, pointing to significant weakening of the P-OAr bond in the transition state. For the metal-catalyzed reactions the data are consistent with a concerted process where the P-OAr bond rupture has progressed to the extent of 84% in the La3+ reaction and ca. 70% in the Zn2+ catalyzed reaction. The catalysis afforded by the metal ions is remarkable, being about 10(6)-fold and 10(8)-fold for poor and good leaving groups, respectively, relative to the background reactions at pH 9.1. Solvent deuterium kinetic isotope studies for two of the substrates promoted by 5:Zn2+(-OCH3) give kH/kD = 1.0 +/- 0.1, consistent with a nucleophilic mechanism. A unified mechanism for the metal-catalyzed reactions is presented which involves pre-equilibrium coordination of the substrate to the metal ion followed by intramolecular delivery of a coordinated methoxide.