Efficient and Selective P‐nitrophenyl‐ester‐hydrolyzing Antibodies Elicited by a P‐nitrobenzyl Phosphonate Hapten (original) (raw)

Simple method for selecting catalytic monoclonal antibodies that exhibit turnover and specificity

Biochemistry, 1990

Monoclonal antibodies were raised against a mono-p-nitrophenyl phosphonate ester to elicit catalytic antibodies capable of hydrolyzing the analogous p-nitrophenyl ester or carbonate. Potential catalytic antibody producing clones were selected, by use of a competitive inhibition assay, on the basis of their affinity for a "short" transition-state analogue, a truncated hapten which maximizes the relative contribution of the transition-state structural elements to binding. Of 30-40 clones that would have been examined on the basis of hapten binding alone, 7 were selected and 4 of these catalyzed the hydrolysis of the relevant p-nitrophenyl ester. This competitive inhibition technique represents a general approach for selecting potential catalytic antibodies and significantly increases the probability of obtaining efficient catalytic monoclonal antibodies. Further study of the catalytic antibodies revealed significant rate enhancement (kcat/kuncat-lo4) and substrate specificity for the hydrolysis of the analogous ester and, for three of the antibodies, of the analogous carbonate. The antibodies displayed turnover, an essential feature of enzymes. Evidence that catalysis occurred at the antibody combining sites was provided by the identity of the binding and the catalysis-Inhibition specificity patterns.

Enantioselective hydrolysis of naproxen ethyl ester catalyzed by polyclonal antibodies

Chinese Science Bulletin, 1997

BASED on the hypothesis of ~a u l i n~[ ' ] on enzyme action that free energy difference between the ground and transition state is reduced by the specific binding of active center of enzyme to the intermediate, a large number of antibodies with catalytic power have been obtained successively by using designed molecules (mimicking the stereoelectronic features of the transition state for the reactions) as haptens conjugated to carrier proteins to immunize the animals and screen out the desired monoclonal antibodies through hy bridoma technology[2' 31. The catalytic antibodies which combined the tremendous diversity of antibodies with catalytic power of enzyme opened new horizons of chemical and immunological fields. Antibodies that catalyze a variety of additional reactions, including all types of reactions catalyzed by enzyme, for example,

Regioselective nitration of phenol induced by catalytic antibodies

Journal of protein chemistry, 2002

Catalytic antibodies with a metalloporphyrin cofactor represent a new generation of biocatalysts tailored for selective oxidations. Thus monoclonal antibodies, 3A3, were raised against microperoxidase 8 (MP8), and the corresponding 3A3-MP8 complexes were shown previously to have a high peroxidase activity. This paper shows that those complexes also catalyzed efficiently the nitration of phenol into 2- and 4-nitrophenol by NO2- in the presence of H2O2. pH dependence studies suggested that no amino acid from the antibody protein participated in the heterolytic cleavage of the O-O bond of H2O2. The inhibition of the reaction by cyanide and radical scavengers suggested a MP8-mediated peroxidase-like mechanism, involving the reduction of high-valent iron-oxo species by NO2- and phenol producing, respectively, NO2* and phenoxy radicals, which then reacted to give nitrophenols. Finally, the antibody protein appears to have two major roles: (i) it protects MP8 toward oxidative degradations ...

Mechanistic insights for β-cyclodextrin catalyzed phosphodiester hydrolysis

Journal of Molecular Modeling, 2014

Hydrolysis of phosphodiester bond in different substrates containing alkyl or aryl substituents, in the presence of β-cyclodextrin (β-CD) as a catalyst, has been investigated employing the density functional theory. It has been shown that the mechanism of β-CD catalyzed phosphodiester hydrolysis in modeled substrates viz. [p-nitrophenyl][(2,2) methylpropan] phosphodiester (G1); [p-nitrophenyl] [(2,2)methyl butan] phosphodiester (G2); (p-nitrophenyl) (2methyl pentan) phosphodiester (G3); (p-nitrophenyl) (phenyl) phosphodiester (G4); (p-nitrophenyl) (m-tert-butyl phenyl) phosphodiester (G5) and (p-nitrophenyl) (p-nitrophenyl) phosphodiester (G6) involves net phosphoryl transfer from p-nitrophenyl to the catalyst. The hydrolysis occurs in a single-step D N A N mechanism wherein the β-CD acts as a competitive general base. The nucleophile addition is facilitated via face-to-face hydrogen-bonded interactions from the secondary hydroxyl groups attached to the top rim of β-CD. The insights for cleavage of phosphodiester along the dissociative pathway have been derived using the molecular electrostatic potential studies as a tool. The activation barrier of substrates containing alkyl group (G2 and G3) are found to be lower than those containing aryl groups (G4, G5 and G6).

Phospho-transfer catalysis

Journal of Organometallic Chemistry, 1998

We report here a precise, in situ 31P{1H}-NMR method of assaying enantiopurity of α-hydroxyphosphonate esters, the products of the carbonyl hydrophosphonylation (Pudovik) reaction. This method is based upon a diazaphospholidine chiral derivatising agent (CDA) which satisfies all of the criteria for a precise assay; (i) derivatisation of α-hydroxyphosphonate esters is both rapid and clean, (ii) kinetic resolution is absent and (iii) 31P{1H} chemical shift dispersions are excellent (>5ppm). Calibration of this assay has been achieved by cross-referencing the 31P{1H}-NMR signals obtained for the CDA-derivatised ester of (MeO)2P(O)CHPh(OH) to optical rotation measurements from scalemic material obtained upon lipase catalysed hydrolysis (F–AP 15, Rhizopus oryzae) of (MeO)2P(O)CHPh(OAc). Analysis of NMR chemical shift and coupling parameters for a closely related series of derivatised α-hydroxyphosphonate esters support further configuration assignments on the basis of inference. We report also on the configurational stability of α-hydroxyphosphonate esters in the presence of acids, organonitrogen bases and metal salts. 2H-labelling and carbonyl crossover experiments reveal that low levels of epimerisation (<2%) at the alpha-carbon atom (Cα) of α-hydroxyphosphonate esters is possible under certain conditions of catalysis and within certain limits. A design strategy for the construction of catalyst systems in the Pudovik reaction is outlined based upon a combination of Lewis acidic (E) and Lewis basic (N) sites. Four types of catalyst are outlined, members of two distinct Classes I and II according to the nature of the acid and base sites, along with our investigations of representative examples of each Class. A variety of Class I.1 systems based on β-amino alcohols (one hydrogen bonding E site and one organonitrogen N site), have been assayed in the model reaction between (MeO)2P(O)H and PhCHO. Results suggest that catalysis of the Pudovik reaction is clean and efficient in certain cases but that catalytic activity is strongly dependent upon the nature of the basic (N) nitrogen centre. Moreover, only low levels (<10%) of enantioselectivity are afforded by all amino alcohols assayed. Achiral variants of Class I.2 catalysts (multiple hydrogen bonding E and/or N sites) have been examined to model carbonyl and H-phosphonate binding; an amphoteric receptor based on a pyridine dicarboxamide scaffold has been synthesised and shown to bind benzaldehyde >50% more strongly (K11 0.53 mol−1 dm3) than dimethyl-H-phosphonate (K11 0.34 mol−1 dm3, 298 K) and to catalyse the hydrophosphonylation reaction between these two substrates with a second order rate constant comparable to that of triethylamine (both k2 5.9×10−2 mol−1 dm3 h−1, 293 K). However, one of the major limitations of this model is that competitive product inhibition dominates after some 15 turnovers (75% completion). Model studies reveal that hydrophosphonylation catalysis via a nitrogen Lewis base is accelerated up to 10-fold upon the introduction of [Zn(OSO2CF3)2] as co-catalyst. Consequently, Class II.1 systems employ metal salts [Zn(OSO2CF3)2] as Lewis acidic E sites and chiral co-catalysts capable of binding to the metal and also acting as Lewis basic N sites. Such systems catalyse the addition of (MeO)2P(O)H to PhCHO cleanly with modest turnover numbers (<10 turnovers; 50% completion) but with enhanced enantioselectivity over Class I catalysts (<40% e.e.). However, competitive product inhibition is still problematic. Class II.2 systems are related to Class II.1 but possess directly coordinated E and N sites with more basic N functions and consequently are far more active as catalysts than the other classes. This increased catalytic activity is exemplified by one of the simplest achiral members, diethylzinc which catalyses the 100% chemo- and regioselective addition of (MeO)2P(O)H to PhCHO to afford (MeO)2P(O)CHPh(OH) with an average turnover rate (over a 1 h reaction time at 298 K) of 115 h−1 compared to ca. 1 h−1 for NEt3 under analogous conditions. Chiral variants are proposed.