Gregory Absillis

Direct Observation of Molecular‐Level Template Action Leading to Self‐Assembly of a Porous Framework

The molecular steps involved in the self-assembly of Cu(3)(BTC)(2) (BTC=1,3,5-benzenetricarboxylic acid) metal-organic frameworks that enclose Keggin-type H(3)PW(12)O(40) heteropolyacid molecules were unraveled by using solution (17)O, (31)P, and (183)W NMR spectroscopy, small-angle X-ray scattering, near-IR spectroscopy, and dynamic light scattering. In aqueous solution, complexation of Cu(2+) ions with Keggin-type heteropolyacids was observed. Cu(2+) ions are arranged around the Keggin structure so that linking through benzenetricarboxylate groups results in the formation of the Cu(3)(BTC)(2) MOF structure HKUST-1. This is a unique instance in which a templating mechanism that relies on specific molecular-level matching and leads to explicit nanoscale building units can be observed in situ during formation of the synthetic nanoporous material.

Hydrolytic cleavage of an RNA-model phosphodiester catalyzed by a highly negatively charged polyoxomolybdate [Mo7O24]6- cluster.

Hydrolysis of 2-hydroxypropyl-4-nitrophenyl phosphate (HPNP), a commonly used RNA model substrate, was examined in molybdate solutions by means of (1)H, (31)P, and (95)Mo NMR, Raman, and Mo K-edge extended X-ray absorption fine structure (EXAFS) spectroscopy. (1)H and (31)P NMR spectroscopy indicate that at 50 degrees C and pD 5.9 the cleavage of the phosphodiester bond in HPNP proceeds with a rate constant of 6.62 x 10(-6) s(-1), giving a cyclic phosphate ester and p-nitrophenol as the only products of hydrolysis. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), and indicating that the cleavage of the phosphodiester bond is purely hydrolytic. The Mo K-edge XANES region also did not show any sign of Mo(VI) to Mo(V) reduction during the hydrolytic reaction. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest cleavage observed at pD 5.9. Comparison of the rate profile with the concentration profile of polyoxomolybdates shows a striking overlap of the k(obs) profile with the concentration of heptamolybdate, suggesting that the highly negatively charged [Mo(7)O(24)](6-) is the hydrolytically active species. Kinetic experiments at pD 5.9 using a fixed amount of [Mo(7)O(24)](6-) and increasing amounts of HPNP revealed slight signs of curvature at 25 molar excess of HPNP. The data fit the general Michaelis-Menten reaction scheme, permitting the calculation of the catalytic rate constant k(2) (3.02 x 10(-4) s(-1)) and K(m) (1.06 M). Variable temperature (31)P NMR spectra of a reaction mixture revealed broadening of the HPNP (31)P resonance upon increase of temperature, implying the dynamic exchange process between free and bound HPNP at higher temperatures. Addition of salts resulted in the inhibition of HPNP hydrolysis, as well as addition of dimethyl phosphate, suggesting competition for the binding to [Mo(7)O(24)](6-). The hydrolysis of 10 equiv of HPNP could be achieved in the presence of 1 equiv of [Mo(7)O(24)](6-), and the multiple turnovers demonstrate that the reaction is catalytic. (31)P NMR and Mo K-edge EXAFS spectra measured during different stages of the hydrolysis indicated that under catalytic conditions a partial conversion of [Mo(7)O(24)](6-) into [P(2)Mo(5)O(23)](6-) occurs.

Peptide bond hydrolysis catalyzed by the Wells-Dawson Zr(α2-P2W17O61)2 polyoxometalate.

In this paper we report the first example of peptide hydrolysis catalyzed by a polyoxometalate complex. A series of metal-substituted Wells-Dawson polyoxometalates were synthesized, and their hydrolytic activity toward the peptide bond in glycylglycine (GG) was examined. Among these, the Zr(IV)- and Hf(IV)-substituted ones were the most reactive. Detailed kinetic studies were performed with the Zr(IV)-substituted Wells-Dawson type polyoxometalate K(15)H[Zr(α(2)-P(2)W(17)O(61))(2)]·25H(2)O which was shown to act as a catalyst for the hydrolysis of the peptide bond in GG. The speciation of K(15)H[Zr(α(2)-P(2)W(17)O(61))(2)]·25H(2)O which is highly dependent on the pD, concentration, and temperature of the solution, was fully determined with the help of (31)P NMR spectroscopy and its influence on the GG hydrolysis rate was examined. The highest reaction rate (k(obs) = 9.2 (±0.2) × 10(-5) min(-1)) was observed at pD 5.0 and 60 °C. A 10-fold excess of GG was hydrolyzed in the presence of K(15)H[Zr(α(2)-P(2)W(17)O(61))(2)]·25H(2)O proving the principles of catalysis. (13)C NMR data suggested the coordination of GG to the Zr(IV) center in K(15)H[Zr(α(2)-P(2)W(17)O(61))(2)]·25H(2)O via its N-terminal amine group and amide carbonyl oxygen. These findings were confirmed by the inactivity of K(15)H[Zr(α(2)-P(2)W(17)O(61))(2)]·25H(2)O toward the N-blocked analogue acetamidoglycylglycinate and the inhibitory effect of oxalic, malic, and citric acid. Triglycine, tetraglycine, and pentaglycine were also fully hydrolyzed in the presence of K(15)H[Zr(α(2)-P(2)W(17)O(61))(2)]·25H(2)O yielding glycine as the final product of hydrolysis. K(15)H[Zr(α(2)-P(2)W(17)O(61))(2)]·25H(2)O also exhibited hydrolytic activity toward a series of other dipeptides.

Regioselective Hydrolysis of Human Serum Albumin by ZrIV-Substituted Polyoxotungstates at the Interface of Positively Charged Protein Surface Patches and Negatively Charged Amino Acid Residues

Complexes comprising the Lewis acidic Zr(IV) metal and protein binding polyoxotungstate ligands of Lindqvist-, Keggin- and Wells-Dawson-type were found to region selectively hydrolyze human serum albumin at four distinct positions. Higher reactivities were found for structures with higher polyoxometalate charges and the cleavage positions were found in protein regions of mixed charge. Both findings suggest an electrostatic nature of the observed reactivity.

Questioning the paradigm of metal complex promoted phosphodiester hydrolysis: [Mo7O24](6-) polyoxometalate cluster as an unlikely catalyst for the hydrolysis of a DNA model substrate.

The first example of a phosphodiester bond cleavage promoted by a highly negatively charged polyoxometalate cluster has been discovered: the hydrolysis of the phosphodiester bond in a DNA model substrate bis(p-nitrophenyl)phosphate (BNPP) is promoted by the heptamolybdate anion [Mo7O24](6-) with rates which represent an acceleration of nearly four orders of magnitude compared to the uncatalyzed cleavage.

Hydrolysis of DNA model substrates catalyzed by metal-substituted Wells–Dawson polyoxometalates

In this study we report the first example of phosphoester bond hydrolysis in 4-nitrophenyl phosphate (NPP) and bis-4-nitrophenyl phosphate (BNPP), two commonly used DNA model substrates, promoted by metal-substituted polyoxometalates (POMs). Different transition metal and lanthanide ions were incorporated into the Wells-Dawson polyoxometalate framework and subsequently screened for their hydrolytic activity towards the cleavage of the phosphoester bonds in NPP and BNPP. From these complexes, the Zr(iv)-substituted POM showed the highest reactivity. At pD 7.2 and 50 °C a NPP hydrolysis rate constant of 7.71 × 10(-4) min(-1) (t(1/2) = 15 h) was calculated, representing a rate enhancement of nearly two orders of magnitude in comparison with the spontaneous hydrolysis of NPP. The catalytic (k(c) = 1.73 × 10(-3) min(-1)) and formation constant (K(f) = 520.02 M(-1)) for the NPP-Zr(iv)-POM complex were determined from kinetic experiments. The reaction proceeded faster in acidic conditions and (31)P NMR experiments showed that faster hydrolysis is proportional to the presence of the 1 : 1 monosubstituted Zr(iv)-POM at acidic pD values. The strong interaction of the 1 : 1 monosubstituted Zr(iv)-POM with the P-O bond of NPP was evidenced by the large chemical shift and the line broadening of the (31)P nucleus in NPP observed upon addition of the metal complex. Significantly, a ten-fold excess of NPP was fully hydrolyzed in the presence of the Zr(iv)-POM, proving the principles of catalysis. The NMR spectra did not show sign of any paramagnetic species, excluding an oxidative cleavage mechanism and suggesting purely hydrolytic cleavage.

Phosphoesterase activity of polyoxomolybdates: diffusion ordered NMR spectroscopy as a tool for obtaining insights into the reactivity of polyoxometalate clusters

Diffusion ordered NMR spectroscopy (DOSY NMR) is shown to be an excellent tool for observing reactive transients in the hydrolysis of the phosphatase model substrate (p-nitrophenyl)phosphate (NPP) promoted by polyoxomolybdate.

Molecular interactions between serum albumin proteins and Keggin type polyoxometalates studied using luminescence spectroscopy.

The interaction between the plenary Keggin H3PW12O40, lacunary Keggin K7PW11O39 and the Eu(III)-substituted Keggin K4EuPW11O39 (Eu-Keggin) type polyoxometalates (POMs), and the proteins human and bovine serum albumin (HSA and BSA) was studied using steady state and time-resolved Eu(III) luminescence and tryptophan (Trp) fluorescence spectroscopy. The excitation spectrum of the Eu-Keggin POM is dominated by a ligand-to-metal charge transfer band at 291 nm. In the absence of proteins, the number of water molecules coordinated in the first coordination sphere of the Eu(III) center of Eu-Keggin was determined to be 4, indicating that Eu(III) occurs as a 1 : 1 isomer in solution. In the presence of HSA or BSA, the number of coordinated water molecules decreased to 0 and 1, respectively, suggesting interaction between the Eu-Keggin POM and the protein surface. As a result of this interaction, a five-fold increase of the hypersensitive (5)D0 → (7)F2 transition in the luminescence intensity was observed for the Eu-Keggin-HSA complex. The association constants were calculated to be 1.5 × 10(2) M(-1) and 2.0 × 10(3) M(-1) for the Eu-Keggin-HSA and Eu-Keggin-BSA complexes, respectively. Tryptophan fluorescence quenching studies were performed and the quenching constants were calculated using a Stern-Volmer analysis. The obtained values of the quenching constants were 6.1 × 10(4) M(-1) and 2.0 × 10(6) M(-1) for the Eu-Keggin-HSA and Eu-Keggin-BSA complexes, respectively. The surface map of both proteins shows that the cavity containing the tryptophan has a positive surface potential, providing a specific binding site at the surface of albumin proteins for the negatively charged POM.

Polyoxomolybdate Promoted Hydrolysis of a DNA-Model Phosphoester Studied by NMR and EXAFS Spectroscopy

Hydrolysis of (p-nitrophenyl)phosphate (NPP), a commonly used phosphatase model substrate, was examined in molybdate solutions by means of (1)H, (31)P, and (95)Mo NMR spectroscopy and Mo K-edge Extended X-ray Absorption Fine Structure (EXAFS) spectroscopy. At 50 °C and pD 5.1 the cleavage of the phosphoester bond in NPP proceeds with a rate constant of 2.73 × 10(-5) s(-1) representing an acceleration of nearly 3 orders of magnitude as compared to the hydrolysis measured in the absence of molybdate. The pD dependence of k(obs) exhibits a bell-shaped profile, with the fastest cleavage observed in solutions where [Mo(7)O(24)](6-) is the major species in solution. Mixing of NPP and [Mo(7)O(24)](6-) resulted in formation of these two intermediate complexes that were detected by (31)P NMR spectroscopy. Complex A was characterized by a (31)P NMR resonance at -4.27 ppm and complex B was characterized by a (31)P NMR resonance at -7.42 ppm. On the basis of the previous results from diffusion ordered NMR spectroscopy, performed with the hydrolytically inactive substrate phenylphosphonate (PhP), the structure of these two complexes was deduced to be (NPP)(2)Mo(5)O(21)(4-) (complex A) and (NPP)(2)Mo(12)O(36)(H(2)O)(6)(4-) (complex B). The pH studies point out that both complexes are hydrolytically active and lead to the hydrolysis of phosphoester bond in NPP. The NMR spectra did not show evidence of any paramagnetic species, excluding the possibility of Mo(VI) reduction to Mo(V), and indicating that the cleavage of the phosphomonoester bond is purely hydrolytic. The Mo K-edge XANES region also did not show any sign of Mo(VI) to Mo(V) reduction during the hydrolytic reaction. (95)Mo NMR and Mo K-edge EXAFS spectra measured during different stages of the hydrolytic reaction showed a gradual disappearance of [Mo(7)O(24)](6-) during the hydrolytic reaction and appearance of [P(2)Mo(5)O(23)](6-), which was the final complex observed at the end of hydrolytic reaction.

Amide bond hydrolysis in peptides and cyclic peptides catalyzed by a dimeric Zr(IV)-substituted Keggin type polyoxometalate.

Detailed kinetic studies on the hydrolysis of glycylserine (Gly-Ser) and glycylglycine (Gly-Gly) in the presence of the dimeric zirconium(IV)-substituted Keggin type polyoxometalate (Et2NH2)8[{α-PW11O39Zr(μ-OH)(H2O)}2]·7H2O (1) were performed by a combination of (1)H, (13)C and (31)P NMR spectroscopy. The observed rate constants for the hydrolysis of Gly-Ser and Gly-Gly at pD 5.4 and 60 °C were 63.3 × 10(-7) s(-1) and 4.44 × 10(-7) s(-1) respectively, representing a significant acceleration as compared to the uncatalyzed reactions. The pD dependence of the rate constant for both reactions exhibited a bell-shaped profile with the fastest hydrolysis observed in the pD range of 5.5-6.0. Interaction of 1 with Gly-Ser and Gly-Gly via their amine nitrogen and amide oxygen was proven by (13)C NMR spectroscopy. The effective hydrolysis of Gly-Ser in the presence of 1 is most likely a combination of the polarization of the amide oxygen due to its binding to the Zr(IV) ion in 1 and the intramolecular attack of the Ser hydroxyl group on the amide carbonyl carbon. The effect of temperature, inhibitors, and ionic strength on the hydrolysis rate constant was also examined. The solution structure of 1 was investigated by means of (31)P NMR spectroscopy, revealing that its stability is highly dependent on pH, concentration and temperature. A 2.0 mM solution of 1 was found to be fully stable under hydrolytic conditions (pD 5.4 and 60 °C) both in the presence and in the absence of the dipeptides.