Hydrolysis of 4-nitrophenyl acetate by a (N2S(thiolate))zinc hydroxide complex: a model of the catalytically active intermediate for the zinc form of peptide deformylase

A novel zinc(II) hydroxide complex with a rare alkylthiolate donor in the coordination sphere is formed in aqueous solution from the dissolution of the zinc alkyl precursor complex (PATH)ZnCH(3) (PATH = 2-methyl-1-[methyl(2-pyridin-2-ylethyl)amino]propane-2-thiolate) in H(2)O and protonolysis of the Zn-C bond to give (PATH)ZnOH (1). The (PATH)ZnOH complex has been shown to promote the hydrolysis of 4-nitrophenyl acetate (4-NA) by a detailed kinetic study and is the first functional model for the zinc form of the enzyme peptide deformylase. From a fit of the sigmoidal pH-rate profile a kinetic pK(a) of 8.05(5) and a pH-independent second-order rate constant (k" max)) of 0.089(3) M(-1) s(-1) have been obtained. The kinetic pK(a) is similar to the pK(a) of 7.7(1) determined by a potentiometric study (25 degrees C, I = 0.1 (NaNO3)). Observation of both rate enhancement and turnover shows that 1 acts as a catalyst for the hydrolysis of 4-NA, although the turnovers are modest. Activation parameters have been obtained from a temperature-dependence study of the rate constants (E(a) = 54.8 kJ mol(-1), DeltaH++ = 52.4 kJ mol(-1), and DeltaS++ = -90.0 J mol(-1) K(-1)), and support a reaction mechanism which depends on nucleophilic attack of 1 in the rate-determining step. This is the first kinetic and thermodynamic study of a 4-coordinate zinc hydroxide complex containing a thiolate donor. In addition it is only the second time that a complete set of activation parameters have been obtained for the zinc-promoted hydrolysis of a carboxylic ester. This study puts the basicity and nucleophilicity of a (N(2)S)ZnOH complex in context with those of other L(n)()ZnOH complexes and enzymes.     

Amide hydrolysis reactivity of a N4O-ligated zinc complex: comparison of kinetic and themodynamic parameters with those of the corresponding amide methanolysis reaction

Treatment of the mononuclear amide-appended zinc complex [(ppbpa)Zn](ClO4)2 (1(ClO4)2) with Me4NOH.5H2O in CD3CN/D2O (3:1) results in the formation of the deprotonated amide species [(ppbpa-)Zn]ClO4 (2). Upon heating in CD3CN/D2O, this complex undergoes amide hydrolysis to produce a zinc carboxylate product, [(ambpa)Zn(O2CC(CH3)3)]ClO4 (3). X-ray crystallography, 1H and 13C NMR, IR, and elemental analysis were used to characterize 3. The hydrolysis reaction of 1(ClO4)2 exhibits saturation kinetic behavior with respect to the concentration of D2O. Variable-temperature kinetic studies of the amide hydrolysis reaction yielded DeltaH++ = 18.0(5) kcal/mol and DeltaS++ = -22(2) eu. These activation parameters are compared to those of the corresponding amide methanolysis reaction of 1(ClO4)2.     

Ester hydrolysis by a cyclodextrin dimer catalyst with a metallophenanthroline linking group

A novel beta-cyclodextrin dimer, 1,10-phenanthroline-2,9-dimethyl-bridged-bis(6-monoammonio-beta-cyclodextrin) (phenBisCD, L), was synthesized. Its zinc complex (ZnL) has been prepared, characterized, and applied as a new catalyst for diester hydrolysis. The formation constant (logK(ML)=9.56+/-0.01) of the complex and deprotonation constant (pK(a)=8.18+/-0.04) of the coordinated water molecule were determined by a potentiometric pH titration at (298+/-0.1) K. Hydrolytic kinetics of carboxylic acid esters were performed with bis(4-nitrophenyl) carbonate (BNPC) and 4-nitrophenyl acetate (NA) as substrates. The obtained hydrolysis rate constants showed that ZnL has a very high rate of catalysis for BNPC hydrolysis, giving a 3.89x10(4)-fold rate enhancement over uncatalyzed hydrolysis at pH 7.01, relative to only a 42-fold rate enhancement for NA hydrolysis. Moreover, the hydrolysis second-order rate constants of both BNPC and NA greatly increases with pH. Hydrolytic kinetics of a phosphate diester catalyzed by ZnL was also investigated by using bis(4-nitrophenyl) phosphate (BNPP) as the substrate. The pH dependence of the BNPP cleavage in aqueous buffer shows a sigmoidal curve with an inflection point around pH 8.11, which was nearly identical to the pK(a) value from the potentiometric titration. The k(cat) of BNPP hydrolysis promoted by ZnL was found to be 9.9x10(-4) M(-1) s(-1), which is comparatively higher than most other reported Zn(II)-based systems. The possible intermediate for the hydrolysis of BNPP, BNPC, and NA catalyzed by ZnL is proposed on the basis of kinetic and thermodynamic analysis.     

Kinetics and mechanism of hydrolysis of a model phosphate diester by [Cu(Me3tacn)(OH2)2]2+ (Me3tacn = 1,4,7-trimethyl-1,4,7-triazacyclononane)

The kinetics of hydrolysis of bis(p-nitrophenyl)phosphate (BNPP) by [Cu(Me3tacn)(OH2)2]2+ has been studied by spectrophotometrical monitoring of the release of the p-nitrophenylate ion from BNPP. The reaction was followed for up to 8000 min at constant BNPP concentration (15 microM) and ionic strength (0.15 M) and variable concentration of complex (1.0-7.5 mM) and temperature (42.5-65.0 degrees C). Biphasic kinetic traces were observed, indicating that the complex promotes the cleavage of BNPP to NPP [(p-nitrophenyl)phosphate] and then cleavage of the latter to phosphate, the two processes differing in rate by 50-100-fold. Analysis of the more amenable cleavage of BNPP revealed that the rate of BNPP cleavage is among the highest measured for mononuclear copper(II) complexes and is slightly higher than that reported for the close analogue [Cu(iPr3tacn)(OH2)2]2+. Detailed analysis required the determination of the pKa for [Cu(Me3tacn)(OH2)2]2+ and the constant for the dimerization of the conjugate base to [(Me3tacn)Cu(OH)2Cu(Me3tacn)]2+ (Kdim). Thermodynamic parameters derived from spectrophotometric pH titration and the analysis of the kinetic data were in reasonable agreement. Second-order rate constants for cleavage of BNPP by [Cu(Me3tacn)(OH2)(OH)]+ and associated activation parameters were obtained from initial rate analysis (k = 0.065 M(-1) s(-1) at 50.0 degrees C, deltaH = 56+/-6 kJ mol(-1), deltaS = -95+/-18 J K(-1) mol(-1)) and biphasic kinetic analysis (k = 0.14 M(-1) s(-1) at 50.0 degrees C, deltaH = 55+/-6 kJ mol(-1), deltaS = -92+/-20 J K(-1) mol(-1)). The negative entropy of activation is consistent with a concerted mechanism with considerable associative character. The complex was found to catalyze the cleavage of BNPP with turnover rates of up to 1 per day. Although these turnover rates can be considered low from an application point of view, the ability of the complexes to catalyze phosphate ester cleavage is clearly demonstrated.     

A fluorescent probe for zinc ions based on N-methyltetraphenylporphine with high selectivity

N-methyl-alpha,beta,gamma,delta-tetraphenylporphine (NMTPPH) has been used to detect trace amount of zinc ions in ethanol-water solution by fluorescence spectroscopy. The fluorescent probe undergoes a fluorescent emission intensity enhancement upon binding to zinc ions in EtOH/H(2)O (1:1, v/v) solution. The fluorescence enhancement of NMTPPH is attributed to the 1:1 complex formation between NMTPPH and Zn(II) which has been utilized as the basis for the selective detection of Zn(II). The linear response range covers a concentration range of Zn(II) from 5.0x10(-7) to 1.0x10(-5)mol/L and the detection limit is 1.5x10(-7)mol/L. The fluorescent probe exhibits high selectivity over other common metal ions except for Cu(II).     

Phosphodiester cleavage of ribonucleoside monophosphates and polyribonucleotides by homo- and heterodinuclear metal complexes of a cyclohexane-based polyamino-polyol ligand

The ability of the dinuclear complexes of tdci [1,3,5-trideoxy-1,3,5-tris(dimethylamino)-cis-inositol] to promote the cleavage of the phosphodiester bonds of nucleoside 2',3'-cyclic monophosphates, dinucleoside monophosphates and polyribonucleotides has been studied. The homodinuclear copper(II) and zinc(II) complexes efficiently promote the hydrolysis of cyclic nucleotides. The second-order rate constant (k(2) approximately 0.44 M(-1) s(-1)) estimated for the cleavage of 2',3'-cAMP induced by dinuclear copper(II) complexes is about 107 times greater than that for the hydroxide-ion-catalysed reaction. The complex selectively cleaves the 2'O-P bond of 2',3'-cUMP and forms the 3'-product in 91 % yield. An equimolar mixture of copper(II), zinc(II) and tdci proved to be more efficient than either of the binary systems: a 7-20-fold rate enhancement was observed for the cleavage of 2',3'-cNMP substrates. The half-life for the hydrolysis of 2',3'-cAMP decreased from 300 days to five minutes at 25 degrees C when the concentration of each of the three components was 2.5 mM. In contrast to the copper(II) or zinc(II) complexes of tdci, the heterodinuclear species promoted the hydrolysis of several dinucleoside monophosphates. For two ApA isomers, cleavage of the 3',5'-bond was about 6.5 times faster than cleavage of the 2',5'-bond. On the basis of the kinetic data, a trifunctional mechanism is suggested for the heterodinuclear-complex-promoted cleavage of the phosphodiester bond. Double Lewis acid activation occurs when the metal ions bind to the phosphate oxygen atoms. In particular, a metal-bound hydroxide ion serves as a general base or a nucleophilic catalyst, and, presumably, a zinc(II)-bound aqua ligand behaves as a general acid and facilitates the departure of the leaving alkoxide group. The effect of the complexes on the hydrolysis of poly(U), poly(A) and type III native RNA was also investigated, and, for the first time, kinetic data on the cleavage of the phosphodiester bonds of polyribonucleotides by a dinuclear complex was obtained.     

Hydrolysis of p-nitrophenyl picolinate catalyzed by metal complexes of N-alkyl-3,5-bis(hydroxymethyl)-1,2,4-triazole in CTAB micelles

Two lipophilic ligands containing triazole and hydroxyl groups, N-alkyl(C(n)H(2n+1))-3,5-bis(hydroxymethyl)-1,2,4-triazole (n=10 and 12), were synthesized. Effects of their Cu(II) and Ni(II) complexes on the hydrolysis of p-nitrophenyl picolinate (PNPP) in cetyltrimethylammonium bromide (CTAB) micelles have been investigated kinetically, and some kinetic parameters of the reactions were obtained by employing the ternary complex kinetic model for metallomicellar catalysis. It was found that Cu(II) complexes of these triazole-based ligands showed more effective catalytic activity on the hydrolysis of PNPP than Ni(II) complexes. Also, the apparent first-order rate constants for product formation in the metallomicellar phase (k(N)(')), the association constants between the substrate and the binary complex (K(T)), and the association constants between the metal ion and the ligand (K(M)) increased with an increase in pH value, which may be attributed to an increase in the nucleophilicity of the hydroxyl groups in the ligand or the electrophilicity of the substrate at higher pH. In addition, at constant pH, k(N)(') and K(T) increased with an increase in the hydrocarbon chain length of the ligand, while K(M) decreased.     

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex

Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex (Zn2L3+) has been studied (L = alkoxide species of 1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-ol). Potentiometric pH titration study disclosed a 1 : 1 phenyl phosphate complexation with Zn2L3+ in aqueous solution. The dissociation constant (= [Zn2L3+][PhOPO3(2-)]/[Zn2L3+-PhOPO3(2-)]) is an extremely small value of 2.5 x 10(-8) mol dm(-3) at 25 degrees C with I = 0.10 (NaNO3). The X-ray crystal analysis of the dizinc(II) complex with p-nitrophenyl phosphate showed that the phosphate dianion binds as a bridging ligand to the two zinc(II) ions.     

Solvent induced cooperativity of Zn(II) complexes cleaving a phosphate diester RNA analog in methanol

The kinetics of cyclization of 2-hydroxypropyl p-nitrophenyl phosphate (1) promoted by two mononuclear Zn(II) catalytic complexes of bis(2-pyridylmethyl)benzylamine (4) and bis(2-methyl 6-pyridylmethyl)benzylamine (5) in methanol were studied under (s)(s)pH-controlled conditions (where (s)(s)pH refers to [H(+)] activity in methanol). Potentiometric titrations of the ligands in the absence and presence of Zn(2+) and a non-reactive model for 1 (2-hydroxylpropyl isopropyl phosphate (HPIPP, 6)) indicate that the phosphate is bound tightly to the 4:Zn(II) and 5:Zn(II) complexes as L:Zn(II):6(-), and that each of these undergoes an additional ionization to produce L:Zn(II):6(-):((-)OCH(3)) or a bound deprotonated form of the phosphate, L:Zn(II):6(2-). Kinetic studies as a function of [L:Zn(II)] indicate that the rate is linear in [L:Zn(II)] at concentrations well above those required for complete binding of the substrate. Plots of the second order rate constants (defined as the gradient of the rate constant vs. [complex] plot) vs. (s)(s)pH in methanol are bell-shaped with rate maxima of 23 dm mol(-1) s(-1) and 146 dm mol(-1) s(-1) for 4:Zn(II) and 5:Zn(II), respectively, at their (s)(s)pH maxima of 10.5 and 10. A mechanism is proposed that involves binding of one molecule of complex to the phosphate to yield a poorly reactive 1 : 1 complex, which associates with a second molecule of complex to produce a transient cooperative 2 : 1 complex within which the cyclization of 1 is rapid. The observations support an effect of the reduced polarity solvent that encourages the cooperative association of phosphate and two independent mononuclear complexes to give a reactive entity.     

Copper(II) complexes of novel N-alkylated derivatives of cis,cis-1,3,5-triaminocyclohexane. 2. Metal-promoted phosphate diester hydrolysis

Aqueous copper(II) N,N',N' '-trimethyl-cis,cis-1,3,5-triaminocyclohexane (Cu(tach-Me(3))(2+)(aq)) promotes the hydrolysis of activated phosphate diesters in aqueous medium at pH 7.2. This complex is selective for cleavage of the phosphate diester sodium bis(p-nitrophenyl) phosphate (BNPP), the rate of hydrolysis of the monoester disodium p-nitrophenyl phosphate being 1000 times slower. The observed rate acceleration of BNPP hydrolysis is slightly greater than that observed for other Cu(II) complexes, such as [Cu([9]aneN(3))Cl(2)] ([9]aneN(3) identical with 1,4,7-triazacyclononane). The rate of hydrolysis is first-order in phosphate ester at low ester concentration and second-order in [Cu(tach-Me(3))](2+)(aq), suggesting the involvement of two metal complexes in the mechanism of substrate hydrolysis. The reaction exhibits saturation kinetics with respect to BNPP concentration according to a modified Michaelis-Menten mechanism: 2CuL + S <==> LCu-S-CuL --> 2CuL + products (K(M) = 12.3 +/- 1.8 mM(2), k(cat) = (4.0 +/- 0.4) x 10(-)(4) s(-1), 50 degrees C) where CuL (triple bond) [Cu(tach-Me(3))](2+), S (triple bond) BNPP, and LCu-S-CuL is a substrate-bridged dinuclear complex. EPR data indicate that the dicopper complex is formed only in the presence of BNPP; the active LCu-S-CuL intermediate species then slowly decays to products, regenerating monomeric CuL.     

Extraction equilibria of some transition metal ions by bis(2-ethylhexyl)phosphinic acid

Measurements of dimerization constants (K(d,HR)) and distribution constants (K(D,HR)) of bis(2-ethylhexyl)phosphinic acid (PIA-8) in three kinds of organic diluents were carried out by a potentiometric two-phase titration technique at 298 +/- 0.1 K. Extraction of iron(III), zinc(II), copper(II), manganese(II), cadmium(II), cobalt(II) and nickel(II) by PIA-8 from 1.0 mol dm(-3) ammonium sulfate solution into heptane was investigated as a function of pH and extractant concentration. The data have been analyzed both graphically and numerically to determine the stoichiometry of extracted species and their extraction constants. The extracted metal species were found to be FeR(3) . 2HR for iron(III), ZnR(2) and ZnR(2) . 3HR for zinc(II), CuR(2) . HR and CuR(2) . 5HR for copper(II), MnR(2) . 2HR and MnR(2) . 3HR for manganese(II), CdR(2) . 3HR for cadmium(II), CoR(2) . HR and CoR(2) . 4HR for cobalt(II) and NiR(2) . 3HR and NiR(2) . 6HR for nickel(II), respectively.     

Metallomicellar catalysis: hydrolysis of phosphate monoester and phosphodiester by Cu(II), Zn(II) complexes of pyridyl ligands in CTAB micellar solution

The catalytic hydrolysis of bis(p-nitrophenyl) phosphate (BNPP) and p-nitrophenyl phosphate (NPP) by metallomicelles composed of Cu(II) or Zn(II) complexes of bispyridine-containing alkanol ligands in CTAB micellar solution was investigated at 30 degrees C. The experimental results indicate that the complexes with a 1:1 ratio of ligands to metal ions for ligands 1 (1,7-bis(6-hydroxymethyl-2-pyridyl)-2,6-dioxaheptane) and 3 (1,4-bis[(6-hydroxymethyl-2-pyridyl)-2-oxapropyl]benzene) and a 1:2 ratio of ligands to metal ions for ligand 2 (1,14-bis(6-hydroxymethyl-2-pyridyl)-2,13-dioxatetradecane) in CATB micellar solution are the active species for the catalytic hydrolysis of BNPP and NPP, respectively. The ternary complex kinetic model for metallomicellar catalysis was employed to obtain the relative kinetic and thermodynamic parameters, which demonstrated the catalytic mechanism for the hydrolysis of BNPP and NPP by metallomicelles.     

1,4,7,10-tetraazacyclododecane metal complexes as potent promoters of phosphodiester hydrolysis under physiological conditions

Previously reported mono- and dinuclear Zn(II), Cu(II), and Ni(II) complexes of 1,4,7,10-tetrazacyclododecane ([12]aneN4 or cyclen) with different heterocyclic spacers (triazine, pyridine) of various lengths (bi- and tripyridine) or an azacrown-pendant have been tested for the hydrolysis of bis(4-nitrophenyl)phosphate (BNPP) under physiological conditions (pH 7-9, 25 degrees C). All Zn(II) complexes promote the hydrolysis of BNPP under physiological conditions, while those of Cu(II) and Ni(II) do not have a significant effect on the hydrolysis reaction. The hydrolysis kinetics in buffered solutions (0.05 M Bis/Tris, TRIS, HEPES, or CHES, I=0.1 M, NaCl) at 25 degrees C were determined by the initial slope method (product conversion<5%). Comparison of the second-order pH-independent rate constants (kBNPP, M(-1) s(-1)) for the mononuclear complexes ZnL1, ZnL3, and ZnL6, which are 6.1x10 (-5), 5.1x10(-5), and 5.7x10(-5), respectively, indicate that the heterocyclic moiety improves the rate of hydrolysis up to six times over the parent Zn([12]aneN4) complex (kBNPP=1.1x10(-5) M(-1) s(-1)). The reactive species is the Zn(II)-OH- complex, in which the Zn(II)-bound OH- acts as a nucleophile. For dinuclear complexes Zn2L2, Zn2L4, and Zn2L5, the rate of reaction is defined by the degree of cooperation between the metal centers, which is determined by the spacer length. Zn2L2 and Zn2L4 possessing shorter spacers are able to hydrolyze BNPP 1 to 2 orders of magnitudes faster than Zn2L5. The second-order rate constants k of Zn2L4 and Zn2L2 at pH 7, 8, and 9 are significantly higher than those of previously reported related complexes. The high BNPP hydrolytic activity may be related to pi-stacking and hydrophobic interactions between the aromatic spacer moieties and the substrate. Complexes Zn2L4 and Zn2L2 show hydrolytic activity at pH 7 and 8, which allows for the hydrolysis of activated phosphate esters under physiological conditions.     

Rapid three-step cleavage of RNA and DNA model systems promoted by a dinuclear Cu(II) complex in methanol. energetic origins of the catalytic efficacy

A dinuclear Cu(II) complex of 1,3-bis-N(1)-(1,5,9-triazacyclododecyl)propane with an associated methoxide (2-Cu(II)(2):(-OCH(3))) was prepared, and its kinetics of reaction with an RNA model (2-hydroxypropyl-p-nitrophenyl phosphate (1, HPNPP)) and two DNA models (methyl p-nitrophenyl phosphate (3) and iso-butyl p-chlorophenyl phosphate (4)) were studied in methanol solution at (s)(s)pH 7.2 +/- 0.2. X-ray diffraction structures of 2-Cu(II)(2):(-OH)(H(2)O)(CF(3)SO(3)-)(3):0.5CH(3)CH(2)OCH(2)CH(3) and 2-Cu(II)(2):(-OH)((C(6)H(5)CH(2)O)(2)PO(2)-)(CF(3)SO(3)-)2 show the mode of coordination of the bridging -OH and H(2)O between the two Cu(II) ions in the first complex and bridging -OH and phosphate groups in the second. The kinetic studies with 1 and 3 reveal some common preliminary steps prior to the chemical one of the catalyzed formation of p-nitrophenol. With 3, and also with the far less reactive substrate (4), two relatively fast events are cleanly observed via stopped-flow kinetics. The first of these is interpreted as a binding step which is linearly dependent on [catalyst] while the second is a unimolecular step independent of [catalyst] proposed to be a rearrangement that forms a doubly Cu(II)-coordinated phosphate. The catalysis of the cleavage of 1 and 3 is very strong, the first-order rate constants for formation of p-nitrophenol from the complex being approximately 0.7 s(-1) and 2.4 x 10(-3) s(-1), respectively. With substrate 3, 2-Cu(II)(2):(-OCH(3)) exhibits Michaelis-Mentin kinetics with a k(cat)/K(M) value of 30 M(-1) s(-1) which is 3.8 x 10(7)-fold greater than the methoxide promoted reaction of 3 (7.9 x 10(-7) M(-1) s(-1)). A free energy calculation indicates that the binding of 2-Cu(II)(2):(-OCH(3)) to the transition states for 1 and 3 cleavage stabilizes them by -21 and -24 kcal/mol, respectively, relative to that of the methoxide promoted reactions. The results are compared with a literature example where the cleavage of 1 in water is promoted by a dinuclear Zn(II) catalyst, and the energetic origins of the exalted catalysis of the 2-Cu(II)(2) and 2-Zn(II)(2) methanol systems are discussed.     

Trinuclear copper(II) complex showing high selectivity for the hydrolysis of 2'-5' over 3'-5' for UpU and 3'-5' over 2'-5' for ApA ribonucleotides

The cooperative action of multiple Cu(II) nuclear centers is shown to be effective and selective in the hydrolysis of 2'-5' and 3'-5' ribonucleotides. Reported herein is the specific catalysis by two trinuclear Cu(II) complexes of L3A and L3B. Pseudo first-order kinetic studies reveal that the L3A trinuclear Cu(II) complex effects hydrolysis of Up(2'-5')U with a rate constant of 28 x 10(-)(4) min(-)(1) and Up(3'-5')U with a rate constant of 0.5 x 10(-)(4) min(-)(1). The hydrolyses of Ap(3'-5')A and Ap(2'-5')A proceed with rate constants of 24 x 10(-)(4) min(-)(1) and 0.5 x 10(-)(4) min(-)(1) respectively. The L3A trinuclear Cu(II) complex demonstrates high specificity for Up(2'-5')U and Ap(3'-5')A. Similar studies with the more rigid L3B trinuclear Cu(II) complex shows no selectivity and yields lower rate constants for hydrolysis. The selectivity observed with the L3A ligand is attributed to the geometry of the ligand-bound diribonucleotide which ultimately dictates the proximity of the attacking hydroxyl and the phosphoester to a Cu(II) center for activation and subsequent hydrolysis.     

Kinetic and thermodynamic characterization of Co(II)-substrate radical pair formation in coenzyme B12-dependent ethanolamine ammonia-lyase in a cryosolvent system by using time-resolved, full-spectrum continuous-wave electron paramagnetic resonance spectroscopy

The formation of the Co(II)-substrate radical pair catalytic intermediate in coenzyme B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) from Salmonella typhimurium has been studied by using time-resolved continuous-wave electron paramagnetic resonance (EPR) spectroscopy in a cryosolvent system. The 41% v/v DMSO/water cryosolvent allows mixing of holoenzyme and substrate, (S)-2-aminopropanol, at 230 K under conditions of kinetic arrest. Temperature step from 230 to 234-248 K initiates the cleavage of the cobalt-carbon bond and the monoexponential rise (rate constant, k(obs) = tau(obs)(-1)) of the EPR-detected Co(II)-substrate radical pair state. The detection deadtime: tau(obs) ratio is reduced by >10(2), relative to millisecond rapid mixing experiments at ambient temperatures. The EPR spectrum acquisition time is 5tau(obs), the approximately 10(2)-fold slower rate of the substrate radical rearrangement reaction relative to k(obs), and the reversible temperature dependence of the amplitude indicate that the Co(II)-substrate radical pair and ternary complex are essentially at equilibrium. The reaction is thus treated as a relaxation to equilibrium by using a linear two-step, three-state mechanism. The intermediate state in this mechanism, the Co(II)-5'-deoxyadenosyl radical pair, is not detected by EPR at signal-to-noise ratios of 10(3), which indicates that the free energy of the Co(II)-5'-deoxyadenosyl radical pair state is >3.3 kcal/mol, relative to the Co(II)-substrate radical pair. Van't Hoff analysis yields DeltaH13 = 10.8 +/- 0.8 kcal/mol and DeltaS13 = 45 +/- 3 cal/mol/K for the transition from the ternary complex to the Co(II)-substrate radical pair state. The free energy difference, DeltaG13, is zero to within one standard deviation over the temperature range 234-248 K. The extrapolated value of DeltaG13 at 298 K is -2.6 +/- 1.2 kcal/mol. The estimated EAL protein-associated contribution to the free energy difference is DeltaG(EAL) = -24 kcal/mol at 240 K, and DeltaH(EAL) = -13 kcal/mol and DeltaS(EAL) = 38 cal/mol/K. The results show that the EAL protein makes both strong enthalpic and entropic contributions to overcome the large, unfavorable cobalt-carbon bond dissociation energy, which biases the reaction in the forward direction of Co-C bond cleavage and Co(II)-substrate radical pair formation.     

Mechanism of di-tert-butylsilylene transfer from a silacyclopropane to an alkene

Kinetic and thermodynamic studies of the reactions of cyclohexene silacyclopropane 1 and monosubstituted alkenes suggested a possible mechanism for di-tert-butylsilylene transfer. The kinetic order in cyclohexene silacyclopropane 1 and cyclohexene were determined to be 1 and -1, respectively. Saturation kinetic behavior in monosubstituted alkene concentration was observed. Competition experiments between substituted styrenes and a deficient amount of di-tert-butylsilylene from 1 correlated well with the Hammett equation and provided a rho value of -0.666 +/- 0.008, using sigma(p) constants. These data supported a two-step mechanism involving reversible di-tert-butylsilylene extrusion from 1, followed by irreversible concerted electrophilic attack of the silylene on the monosubstituted alkene. Eyring activation parameters were found to be DeltaH++ = 22.1 +/- 0.9 kcal.mol(-1) and DeltaS++ = -15 +/- 2 eu. Competition experiments between cycloalkenes and allylbenzene determined cycloalkenes to be more efficient silylene traps (k(rel) =1.3, DeltaDeltaG++ = 0.200 kcal.mol(-1)). A summary of the data resulted in a postulated reaction coordinate diagram. The mechanistic studies enabled rational modification of reaction conditions that improved the synthetic utility of silylene transfer. Removal of the volatile cyclohexene from the reaction mixture into an evacuated headspace led to the formation of previously inaccessible cyclohexene-derived silacyclopropanes.     

Deprotonation of zinc(II)-water and zinc(II)-alcohol and nucleophilicity of the resultant zinc(II) hydroxide and zinc(II) alkoxide in double-functionalized complexes: theoretical studies on models for hydrolytic zinc enzymes

The factors governing the deprotonation ability of zinc(II)-water and zinc(II)-alcohol and nucleophilicity of the resultant zinc(II) hydroxide and zinc(II) alkoxide as complex models for zinc enzymes have been investigated through Hartree-Fock and density-functional theory methods with the 6-311++G(d,p) basis set. Our calculations showed that in these double-functionalized complexes (i.e., zinc complexes having both a zinc(II)-alcohol motif and a zinc(II)-water motif) zinc(II)-alcohol is preferred in deprotonation over zinc(II)-water (i.e., zinc(II)-alcohol has a much lower pK(a) than zinc-coordinated water in the same molecule). Natural bond orbital analysis revealed that zinc(II) alkoxides are more nucleophilic than their respective counterparts zinc(II) hydroxides. The analysis of the transition state in the transformation reaction from zinc(II) hydroxide species to zinc(II) alkoxide species indicates that zinc(II) alkoxides are the preferred deprotonated species not only thermodynamically but also kinetically. Further examination of the proposed mechanisms of the zinc(II) alkoxide-promoted transesterification path and the zinc(II) hydroxide-promoted hydrolysis path revealed the structures of the intermediates and energy diagrams in the reactions. These results, entitled double-functionalized complexes, for the first time, put a firm theoretical foundation of why the zinc(II)-alcoholic OH is a better model for hydrolytic zinc enzymes (having both stronger acidity and better nucleophilicity).     

Reactivity of mu-hydroxodizinc(II) centers in enzymatic catalysis through model studies

The stable dinuclear complex [Zn2(BPAM)(mu-OH)(mu-O2PPh2)](ClO4)2, where BPAN = 2,7-bis[2-(2-pyridylethyl)-aminomethyl]-1,8-naphthyridine, was chosen as a model to investigate the reactivity of (mu-hydroxo)dizinc(II) centers in metallohydrolases. Two reactions, the hydrolysis of phosphodiesters and the hydrolysis of beta-lactams, were studied. These two processes are catalyzed in vivo by zinc(II)-containing enzymes: P1 nucleases and beta-lactamases, respectively. The former catalyzes the hydrolysis of single-stranded DNA and RNA. beta-Lactamases, expressed in many types of pathogenic bacteria, are responsible for the hydrolytic degradation of beta-lactam antibiotic drugs. In the first step of phosphodiester hydrolysis promoted by the dinuclear model complex, the substrate replaces the bridging diphenylphosphinate. The bridging hydroxide serves as a general base to deprotonate water, which acts as a nucleophile in the ensuing hydrolysis. The dinuclear model complex is only 1.8 times more reactive in hydrolyzing phosphodiesters than a mononuclear analogue, Zn(bpta)(OTf)2, where bpta = N,N-bis(2-pyridylmethyl)-tert-butylamine. Hydrolysis of nitrocefin, a beta-lactam antibiotic analogue, catalyzed by [Zn2(BPAN)(mu-OH)(mu-O2PPh2)](ClO4)2 involves monodentate coordination of the substrate via its carboxylate group, followed by nucleophilic attack of the zinc(II)-bound terminal hydroxide at the beta-lactam carbonyl carbon atom. Collapse of the tetrahedral intermediate results in product formation. Mononuclear complexes Zn(cyclen)-(NO3)2 and Zn(bpta)(NO3)2, where cyclen = 1,4,7,10-tetraazacyclododecane, are as reactive in the beta-lactam hydrolysis as the dinuclear complex. Kinetic and mechanistic studies of the phosphodiester and beta-lactam hydrolyses indicate that the bridging hydroxide in [Zn2(BPAN)(mu-OH)(mu-O2PPh2)](ClO4)2 is not very reactive, despite its low pKa value. This low reactivity presumably arises from the two factors. First, the briding hydroxide and coordinated substrate in [Zn2(BPAN)(mu-OH)(substrate)]2+ are not aligned properly to favor nucleophilic attack. Second, the nucleophilicity of the bridging hydroxide is diminished because it is simultaneously bound to the two zinc(II) ions.     

Mechanistic Investigation into the Cleavage of a Phosphomonoester Mediated by a Symmetrical Oxyimine‐Based Macrocyclic Zinc(II) Complex

Density functional calculations are utilized to explore the hydrolysis mechanisms of the phosphomonoester 4‐nitrophenyl phosphate catalyzed by a symmetrical zinc(II) complex. The formation process and properties of the active catalyst are verified. Eight plausible mechanisms are proposed and categorized into three groups. All of the proposed mechanisms, except for Mechanism 7 (see text), are S_N2‐type addition–substitution reaction pathways. Nucleophilic attack at the ortho position occurs in Mechanism 7 with a relatively high reaction barrier. Mechanisms 1 and 2 in the monocatalyst model, Mechanisms 5 to 7 in the sandwich‐dual‐catalyst model, as well as the nucleophilic addition–substitution step in Mechanism 8 are concerted reaction pathways, whereas the rest appear to occur in a stepwise manner. Meanwhile, the explicit solvent model is utilized to consider direct hydrogen bonds and solvation interactions and these results indicate that the added water molecule is involved in the hydrolysis process, but does not change the mechanisms significantly. Mechanism 8, with the lowest reaction barrier, is the most favored reaction pathway of the eight proposed mechanisms, although Mechanisms 1, 4, and 6 are in competition with Mechanism 8. In consideration of the zinc(II) complex concentration, Mechanism 1 is only the predominant reaction pathway at a low zinc(II) complex concentration; Mechanisms 4 and 6 tend to be more competitive with increasing concentration of the zinc(II) complexes, and Mechanism 8 is favored at high zinc(II) complex concentrations. Our calculated results are consistent with, and can be used to systematically interpret, experimental observations. More importantly, insightful suggestions are made regarding the catalyst design and selection of the reaction environment.